Synthesis, spectroscopic, photoluminescence properties and biological evaluation of novel Zn(II) and Al(III) complexes of NOON tetradentate Schiff bases

NASA Astrophysics Data System (ADS)

Abdel Aziz, Ayman A.; Badr, Ibrahim H. A.; El-Sayed, Ibrahim S. A.

2012-11-01

Novel mononuclear Zn(II) and Al(III) complexes were synthesized from the reactions of Zn(OAc)2·2H2O and anhydrous AlCl3 with neutral N2O2 donor tetradentate Schiff bases; N,N'bis(salicylaldehyde)4,5-dimethyl-1,2-phenylenediamine (H2L1) and N,N'bis(salicylaldehyde)4,5-dichloro-1,2-phenylenediamine (H2L2). The new complexes were fully characterized by using micro analyses (CHN), FT-IR, 1H NMR, UV-Vis spectra and thermal analysis. The analytical data have been showed that, the stoichiometry of the complexes is 1:1. Spectroscopic data suggested tetrahedral and square pyramidal geometries for Zn(II) and Al(III) complexes, respectively. The synthesized Zn(II), and Al(III) complexes exhibited intense fluorescence emission in the visible region upon UV-excitation in methylene chloride solution at ambient temperature. This high fluorescence emission was assigned to the strong coordination of the ligands to the small and the highly charged Zn(II) and Al(III) ions. Such strong coordination seems to extend the π-conjugation of the complexes. Thermal analysis measurements indicated that the complexes have good thermal stability. As a potential application the biological activity (e.g., antimicrobial action) of the prepared ligands and complexes was assessed by in-vitro testing of their effect on the growth of various strains of bacteria and fungi.

Evaluation of adsorption properties of sulphurised activated carbon for the effective and economically viable removal of Zn(II) from aqueous solutions.

PubMed

Anoop Krishnan, K; Sreejalekshmi, K G; Vimexen, V; Dev, Vinu V

2016-02-01

The prospective application of sulphurised activated carbon (SAC) as an ecofriendly and cost-effective adsorbent for Zinc(II) removal from aqueous phase is evaluated, with an emphasis on kinetic and isotherm aspects. SAC was prepared from sugarcane bagasse pith obtained from local juice shops in Sree Bhadrakali Devi Temple located at Ooruttukala, Neyyattinkara, Trivandrum, India during annual festive seasons. Activated carbon modified with sulphur containing ligands was opted as the adsorbent to leverage on the affinity of Zn(II) for sulphur. We report batch-adsorption experiments for parameter optimisations aiming at maximum removal of Zn(II) from liquid-phase using SAC. Adsorption of Zn(II) onto SAC was maximum at pH 6.5. For initial concentrations of 25 and 100mgL(-1), maximum of 12.3mgg(-1) (98.2%) and 23.7mgg(-1) (94.8%) of Zn(II) was adsorbed onto SAC at pH 6.5. Kinetic and equilibrium data were best described by pseudo-second-order and Langmuir models, respectively. A maximum adsorption capacity of 147mgg(-1) was obtained for the adsorption of Zn(II) onto SAC from aqueous solutions. The reusability of the spent adsorbent was also determined. Copyright © 2015 Elsevier Inc. All rights reserved.

Design and applications of a clamp for Green Fluorescent Protein with picomolar affinity

DOE PAGES

Hansen, Simon; Stüber, Jakob C.; Ernst, Patrick; ...

2017-11-24

Green fluorescent protein (GFP) fusions are pervasively used to study structures and processes. Specific GFP-binders are thus of great utility for detection, immobilization or manipulation of GFP-fused molecules. We determined structures of two designed ankyrin repeat proteins (DARPins), complexed with GFP, which revealed different but overlapping epitopes. Here in this paper we show a structure-guided design strategy that, by truncation and computational reengineering, led to a stable construct where both can bind simultaneously: by linkage of the two binders, fusion constructs were obtained that “wrap around” GFP, have very high affinities of about 10–30 pM, and extremely slow off-rates. Theymore » can be natively produced in E. coli in very large amounts, and show excellent biophysical properties. Their very high stability and affinity, facile site-directed functionalization at introduced unique lysines or cysteines facilitate many applications. As examples, we present them as tight yet reversible immobilization reagents for surface plasmon resonance, as fluorescently labelled monomeric detection reagents in flow cytometry, as pull-down ligands to selectively enrich GFP fusion proteins from cell extracts, and as affinity column ligands for inexpensive large-scale protein purification. We have thus described a general design strategy to create a “clamp” from two different high-affinity repeat proteins, even if their epitopes overlap.« less

Design and applications of a clamp for Green Fluorescent Protein with picomolar affinity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hansen, Simon; Stüber, Jakob C.; Ernst, Patrick

Green fluorescent protein (GFP) fusions are pervasively used to study structures and processes. Specific GFP-binders are thus of great utility for detection, immobilization or manipulation of GFP-fused molecules. We determined structures of two designed ankyrin repeat proteins (DARPins), complexed with GFP, which revealed different but overlapping epitopes. Here in this paper we show a structure-guided design strategy that, by truncation and computational reengineering, led to a stable construct where both can bind simultaneously: by linkage of the two binders, fusion constructs were obtained that “wrap around” GFP, have very high affinities of about 10–30 pM, and extremely slow off-rates. Theymore » can be natively produced in E. coli in very large amounts, and show excellent biophysical properties. Their very high stability and affinity, facile site-directed functionalization at introduced unique lysines or cysteines facilitate many applications. As examples, we present them as tight yet reversible immobilization reagents for surface plasmon resonance, as fluorescently labelled monomeric detection reagents in flow cytometry, as pull-down ligands to selectively enrich GFP fusion proteins from cell extracts, and as affinity column ligands for inexpensive large-scale protein purification. We have thus described a general design strategy to create a “clamp” from two different high-affinity repeat proteins, even if their epitopes overlap.« less

A Method for Selective Depletion of Zn(II) Ions from Complex Biological Media and Evaluation of Cellular Consequences of Zn(II) Deficiency

PubMed Central

Richardson, Christopher E. R.; Cunden, Lisa S.; Butty, Vincent L.; Nolan, Elizabeth M.; Lippard, Stephen J.; Shoulders, Matthew D.

2018-01-01

We describe the preparation, evaluation, and application of an S100A12 protein-conjugated solid support, hereafter the “A12-resin,” that can remove 99% of Zn(II) from complex biological solutions without significantly perturbing the concentrations of other metal ions. The A12-resin can be applied to selectively deplete Zn(II) from diverse tissue culture media and from other biological fluids, including human serum. To further demonstrate the utility of this approach, we investigated metabolic, transcriptomic, and metallomic responses of HEK293 cells cultured in medium depleted of Zn(II) using S100A12. The resulting data provide insight into how cells respond to acute Zn(II) deficiency. We expect that the A12-resin will facilitate interrogation of disrupted Zn(II) homeostasis in biological settings, uncovering novel roles for Zn(II) in biology. PMID:29334734

Synthesis, spectroscopic, photoluminescence properties and biological evaluation of novel Zn(II) and Al(III) complexes of NOON tetradentate Schiff bases.

PubMed

Abdel Aziz, Ayman A; Badr, Ibrahim H A; El-Sayed, Ibrahim S A

2012-11-01

Novel mononuclear Zn(II) and Al(III) complexes were synthesized from the reactions of Zn(OAc)(2).2H(2)O and anhydrous AlCl(3) with neutral N2O2 donor tetradentate Schiff bases; N,N'bis(salicylaldehyde)4,5-dimethyl-1,2-phenylenediamine (H(2)L(1)) and N,N'bis(salicylaldehyde)4,5-dichloro-1,2-phenylenediamine (H(2)L(2)). The new complexes were fully characterized by using micro analyses (CHN), FT-IR, (1)H NMR, UV-Vis spectra and thermal analysis. The analytical data have been showed that, the stoichiometry of the complexes is 1:1. Spectroscopic data suggested tetrahedral and square pyramidal geometries for Zn(II) and Al(III) complexes, respectively. The synthesized Zn(II), and Al(III) complexes exhibited intense fluorescence emission in the visible region upon UV-excitation in methylene chloride solution at ambient temperature. This high fluorescence emission was assigned to the strong coordination of the ligands to the small and the highly charged Zn(II) and Al(III) ions. Such strong coordination seems to extend the π-conjugation of the complexes. Thermal analysis measurements indicated that the complexes have good thermal stability. As a potential application the biological activity (e.g., antimicrobial action) of the prepared ligands and complexes was assessed by in-vitro testing of their effect on the growth of various strains of bacteria and fungi. Copyright © 2012 Elsevier B.V. All rights reserved.

Fluorescence resonance energy-transfer affects the determination of the affinity between ligand and proteins obtained by fluorescence quenching method

NASA Astrophysics Data System (ADS)

Xiao, Jianbo; Wei, Xinlin; Wang, Yuanfeng; Liu, Chunxi

2009-11-01

The interaction between esculin and serum albumins was investigated to illustrate that the fluorescence resonance energy-transfer (FRET) affects the determination of the binding constants obtained by fluorescence quenching method. The binding constants ( Ka) obtained by the double-logarithm curve for esculin-BSA and esculin-HSA were 1.02 × 10 7 and 2.07 × 10 4 L/mol, respectively. These results from synchronous fluorescence showed that the Tyr and Trp residues of HSA were affected more deeply than those in BSA. The excitation profile of esculin showed that in the presence of BSA and HSA, the S 0 → S 1 transition of esculin ( λexmax≈340 nm) appears, which is similar to the λemmax of BSA and HSA. The critical distance ( R0) between BSA and esculin is higher than that of HSA, which showed that the affinity of esculin and HSA should be higher than that of BSA. After centrifugation, the concentrations of esculin bound to albumins were determined by means of the fluorescence of esculin. It was found that much more esculin was bound to HSA than to BSA. However, the bound models for BSA and HSA are almost the same. The concentration of esculin bound to serum albumin at first decreased with the addition of esculin and then increased. From above results, it can be concluded that the affinity of esculin and HSA should be higher than that of esculin and BSA. This example showed that in the presence of FRET, the binding constants between ligands and proteins based on fluorescence quenching might be deviated.

Effect of Cu(II), Cd(II) and Zn(II) on Pb(II) biosorption by algae Gelidium-derived materials.

PubMed

Vilar, Vítor J P; Botelho, Cidália M S; Boaventura, Rui A R

2008-06-15

Biosorption of Pb(II), Cu(II), Cd(II) and Zn(II) from binary metal solutions onto the algae Gelidium sesquipedale, an algal industrial waste and a waste-based composite material was investigated at pH 5.3, in a batch system. Binary Pb(II)/Cu(II), Pb(II)/Cd(II) and Pb(II)/Zn(II) solutions have been tested. For the same equilibrium concentrations of both metal ions (1 mmol l(-1)), approximately 66, 85 and 86% of the total uptake capacity of the biosorbents is taken by lead ions in the systems Pb(II)/Cu(II), Pb(II)/Cd(II) and Pb(II)/Zn(II), respectively. Two-metal results were fitted to a discrete and a continuous model, showing the inhibition of the primary metal biosorption by the co-cation. The model parameters suggest that Cd(II) and Zn(II) have the same decreasing effect on the Pb(II) uptake capacity. The uptake of Pb(II) was highly sensitive to the presence of Cu(II). From the discrete model it was possible to obtain the Langmuir affinity constant for Pb(II) biosorption. The presence of the co-cations decreases the apparent affinity of Pb(II). The experimental results were successfully fitted by the continuous model, at different pH values, for each biosorbent. The following sequence for the equilibrium affinity constants was found: Pb>Cu>Cd approximately Zn.

Fluorescence resonance energy-transfer affects the determination of the affinity between ligand and proteins obtained by fluorescence quenching method.

PubMed

Xiao, Jianbo; Wei, Xinlin; Wang, Yuanfeng; Liu, Chunxi

2009-11-01

The interaction between esculin and serum albumins was investigated to illustrate that the fluorescence resonance energy-transfer (FRET) affects the determination of the binding constants obtained by fluorescence quenching method. The binding constants (K(a)) obtained by the double-logarithm curve for esculin-BSA and esculin-HSA were 1.02x10(7) and 2.07x10(4)L/mol, respectively. These results from synchronous fluorescence showed that the Tyr and Trp residues of HSA were affected more deeply than those in BSA. The excitation profile of esculin showed that in the presence of BSA and HSA, the S(0)-->S(1) transition of esculin (lambda(ex)(max) approximately 340nm) appears, which is similar to the lambda(em)(max) of BSA and HSA. The critical distance (R(0)) between BSA and esculin is higher than that of HSA, which showed that the affinity of esculin and HSA should be higher than that of BSA. After centrifugation, the concentrations of esculin bound to albumins were determined by means of the fluorescence of esculin. It was found that much more esculin was bound to HSA than to BSA. However, the bound models for BSA and HSA are almost the same. The concentration of esculin bound to serum albumin at first decreased with the addition of esculin and then increased. From above results, it can be concluded that the affinity of esculin and HSA should be higher than that of esculin and BSA. This example showed that in the presence of FRET, the binding constants between ligands and proteins based on fluorescence quenching might be deviated.

8-aminoquinoline functionalized silica nanoparticles: a fluorescent nanosensor for detection of divalent zinc in aqueous and in yeast cell suspension.

PubMed

Rastogi, Shiva K; Pal, Parul; Aston, D Eric; Bitterwolf, Thomas E; Branen, A Larry

2011-05-01

Zinc is one of the most important transition metal of physiological importance, existing primarily as a divalent cation. A number of sensors have been developed for Zn(II) detection. Here, we present a novel fluorescent nanosensor for Zn(II) detection using a derivative of 8-aminoquinoline (N-(quinolin-8-yl)-2-(3 (triethoxysilyl)propylamino)acetamide (QTEPA) grafted on silica nanoparticles (SiNPs). These functionalized SiNPs were used to demonstrate specific detection of Zn(II) in tris-HCl buffer (pH 7.22), in yeast cell (Saccharomyces cerevisiae) suspension, and in tap water. The silane QTEPA, SiNPs and final product were characterized using solution and solid state nuclear magnetic resonance, Fourier transform infrared, ultraviolet-visible absorption spectroscopy, transmission electron microscopy, elemental analysis, thermogravimetric techniques, and fluorescence spectroscopy. The nanosensor shows almost 2.8-fold fluorescence emission enhancement and about 55 nm red-shift upon excitation with 330 ± 5 nm wavelength in presence of 1 μM Zn(II) ions in tris-HCl (pH 7.22). The presence of other metal ions has no observable effect on the sensitivity and selectivity of nanosensor. This sensor selectively detects Zn(II) ions with submicromolar detection to a limit of 0.1 μM. The sensor shows good applicability in the determination of Zn(II) in tris-HCl buffer and yeast cell environment. Further, it shows enhancement in fluorescence intensity in tap water samples.

Fluorescent-responsive synthetic C1b domains of protein kinase Cδ as reporters of specific high-affinity ligand binding.

PubMed

Ohashi, Nami; Nomura, Wataru; Narumi, Tetsuo; Lewin, Nancy E; Itotani, Kyoko; Blumberg, Peter M; Tamamura, Hirokazu

2011-01-19

Protein kinase C (PKC) is a critical cell signaling pathway involved in many disorders such as cancer and Alzheimer-type dementia. To date, evaluation of PKC ligand binding affinity has been performed by competitive studies against radiolabeled probes that are problematic for high-throughput screening. In the present study, we have developed a fluorescent-based binding assay system for identifying ligands that target the PKC ligand binding domain (C1 domain). An environmentally sensitive fluorescent dye (solvatochromic fluorophore), which has been used in multiple applications to assess protein-binding interactions, was inserted in proximity to the binding pocket of a novel PKCδ C1b domain. These resultant fluorescent-labeled δC1b domain analogues underwent a significant change in fluorescent intensity upon ligand binding, and we further demonstrate that the fluorescent δC1b domain analogues can be used to evaluate ligand binding affinity.

A Fluorescent Protein Scaffold for Presenting Structurally Constrained Peptides Provides an Effective Screening System to Identify High Affinity Target-Binding Peptides

PubMed Central

Kadonosono, Tetsuya; Yabe, Etsuri; Furuta, Tadaomi; Yamano, Akihiro; Tsubaki, Takuya; Sekine, Takuya; Kuchimaru, Takahiro; Sakurai, Minoru; Kizaka-Kondoh, Shinae

2014-01-01

Peptides that have high affinity for target molecules on the surface of cancer cells are crucial for the development of targeted cancer therapies. However, unstructured peptides often fail to bind their target molecules with high affinity. To efficiently identify high-affinity target-binding peptides, we have constructed a fluorescent protein scaffold, designated gFPS, in which structurally constrained peptides are integrated at residues K131–L137 of superfolder green fluorescent protein. Molecular dynamics simulation supported the suitability of this site for presentation of exogenous peptides with a constrained structure. gFPS can present 4 to 12 exogenous amino acids without a loss of fluorescence. When gFPSs presenting human epidermal growth factor receptor type 2 (HER2)-targeting peptides were added to the culture medium of HER2-expressing cells, we could easily identify the peptides with high HER2-affinity and -specificity based on gFPS fluorescence. In addition, gFPS could be expressed on the yeast cell surface and applied for a high-throughput screening. These results demonstrate that gFPS has the potential to serve as a powerful tool to improve screening of structurally constrained peptides that have a high target affinity, and suggest that it could expedite the one-step identification of clinically applicable cancer cell-binding peptides. PMID:25084350

Determination of the affinity of drugs toward serum albumin by measurement of the quenching of the intrinsic tryptophan fluorescence of the protein.

PubMed

Epps, D E; Raub, T J; Caiolfa, V; Chiari, A; Zamai, M

1999-01-01

Binding of new chemical entities to serum proteins is an issue confronting pharmaceutical companies during development of potential therapeutic agents. Most drugs bind to the most abundant plasma protein, human serum albumin (HSA), at two major binding sites. Excepting fluorescence spectroscopy, existing methods for assaying drug binding to serum albumin are insensitive to higher-affinity compounds and can be labour-intensive, time-consuming, and usually require compound-specific assays. This led us to examine alternative ways to measure drug-albumin interaction. One method described here uses fluorescence quenching of the single tryptophan (Trp) residue in HSA excited at 295 nm to measure drug-binding affinity. Unfortunately, many compounds absorb, fluoresce, or both, in this UV wavelength region of the spectrum. Several types of binding phenomenon and spectral interference were identified by use of six structurally unrelated compounds and the equations necessary to make corrections mathematically were derived and applied to calculate binding constants accurately. The general cases were: direct quenching of Trp fluorescence by optically transparent ligands with low or high affinities; binding of optically transparent, non-fluorescent ligands to two specific sites where both sites or only one site result in Trp fluorescence quenching; and chromophores whose absorption either overlaps the Trp emission and quenches by energy transfer or absorbs light at the Trp fluorescence excitation wavelength producing absorptive screening as well as fluorescence quenching. Unless identification of the site specificity of drug binding to serum albumin is desired, quenching of the Trp fluorescence of albumin by titration with ligand is a rapid and facile method for determining the binding affinities of drugs for serum albumin.

Analysis of Protein Interactions with Picomolar Binding Affinity by Fluorescence-Detected Sedimentation Velocity

PubMed Central

2014-01-01

The study of high-affinity protein interactions with equilibrium dissociation constants (KD) in the picomolar range is of significant interest in many fields, but the characterization of stoichiometry and free energy of such high-affinity binding can be far from trivial. Analytical ultracentrifugation has long been considered a gold standard in the study of protein interactions but is typically applied to systems with micromolar KD. Here we present a new approach for the study of high-affinity interactions using fluorescence detected sedimentation velocity analytical ultracentrifugation (FDS-SV). Taking full advantage of the large data sets in FDS-SV by direct boundary modeling with sedimentation coefficient distributions c(s), we demonstrate detection and hydrodynamic resolution of protein complexes at low picomolar concentrations. We show how this permits the characterization of the antibody–antigen interactions with low picomolar binding constants, 2 orders of magnitude lower than previously achieved. The strongly size-dependent separation and quantitation by concentration, size, and shape of free and complex species in free solution by FDS-SV has significant potential for studying high-affinity multistep and multicomponent protein assemblies. PMID:24552356

Adsorption of Zn(II) and Cd(II) ions in batch system by using the Eichhornia crassipes.

PubMed

Módenes, A N; Espinoza-Quiñones, F R; Borba, C E; Trigueros, D E G; Lavarda, F L; Abugderah, M M; Kroumov, A D

2011-01-01

In this work, the displacement effects on the sorption capacities of zinc and cadmium ions of the Eichornia crassipes-type biosorbent in batch binary system has been studied. Preliminary single metal sorption experiments were carried out. An improvement on the Zn(II) and Cd(II) ions removal was achieved by working at 30 °C temperature and with non-uniform biosorbent grain sizes. A 60 min equilibrium time was achieved for both Zn(II) and Cd(II) ions. Furthermore, it was found that the overall kinetic data were best described by the pseudo second-order kinetic model. Classical multi-component adsorption isotherms have been tested as well as a modified extended Langmuir isotherm model, showing good agreement with the equilibrium binary data. Around 0.65 mequiv./g maximum metal uptake associated with the E. crassipes biosorbent was attained and the E. crassipes biosorbent has shown higher adsorption affinity for the zinc ions than for the cadmium ones in the binary system.

Rational synthesis of an exceptionally stable Zn(II) metal-organic framework for the highly selective and sensitive detection of picric acid.

PubMed

Hu, Yingli; Ding, Meili; Liu, Xiao-Qin; Sun, Lin-Bing; Jiang, Hai-Long

2016-04-28

Based on an organic ligand involving both carboxylate and tetrazole groups, a chemically stable Zn(II) metal-organic framework has been rationally synthesized and behaves as a fluorescence chemosensor for the highly selective and sensitive detection of picric acid, an extremely hazardous and strong explosive.

Anle138b and related compounds are aggregation specific fluorescence markers and reveal high affinity binding to α-synuclein aggregates.

PubMed

Deeg, Andreas A; Reiner, Anne M; Schmidt, Felix; Schueder, Florian; Ryazanov, Sergey; Ruf, Viktoria C; Giller, Karin; Becker, Stefan; Leonov, Andrei; Griesinger, Christian; Giese, Armin; Zinth, Wolfgang

2015-09-01

Special diphenyl-pyrazole compounds and in particular anle138b were found to reduce the progression of prion and Parkinson's disease in animal models. The therapeutic impact of these compounds was attributed to the modulation of α-synuclein and prion-protein aggregation related to these diseases. Photophysical and photochemical properties of the diphenyl-pyrazole compounds anle138b, anle186b and sery313b and their interaction with monomeric and aggregated α-synuclein were studied by fluorescence techniques. The fluorescence emission of diphenyl-pyrazole is strongly increased upon incubation with α-synuclein fibrils, while no change in fluorescence emission is found when brought in contact with monomeric α-synuclein. This points to a distinct interaction between diphenyl-pyrazole and the fibrillar structure with a high binding affinity (Kd=190±120nM) for anle138b. Several α-synuclein proteins form a hydrophobic binding pocket for the diphenyl-pyrazole compound. A UV-induced dehalogenation reaction was observed for anle138b which is modulated by the hydrophobic environment of the fibrils. Fluorescence of the investigated diphenyl-pyrazole compounds strongly increases upon binding to fibrillar α-synuclein structures. Binding at high affinity occurs to hydrophobic pockets in the fibrils. The observed particular fluorescence properties of the diphenyl-pyrazole molecules open new possibilities for the investigation of the mode of action of these compounds in neurodegenerative diseases. The high binding affinity to aggregates and the strong increase in fluorescence upon binding make the compounds promising fluorescence markers for the analysis of aggregation-dependent epitopes. Copyright © 2015 Elsevier B.V. All rights reserved.

BIOCHEMISTRY OF MOBILE ZINC AND NITRIC OXIDE REVEALED BY FLUORESCENT SENSORS

PubMed Central

Pluth, Michael D.; Tomat, Elisa; Lippard, Stephen J.

2010-01-01

Biologically mobile zinc and nitric oxide (NO) are two prominent examples of inorganic compounds involved in numerous signaling pathways in living systems. In the past decade, a synergy of regulation, signaling, and translocation of these two species has emerged in several areas of human physiology, providing additional incentive for developing adequate detection systems for Zn(II) ions and NO in biological specimens. Fluorescent probes for both of these bioinorganic analytes provide excellent tools for their detection, with high spatial and temporal resolution. We review the most widely used fluorescent sensors for biological zinc and nitric oxide, together with promising new developments and unmet needs of contemporary Zn(II) and NO biological imaging. The interplay between zinc and nitric oxide in the nervous, cardiovascular, and immune systems is highlighted to illustrate the contributions of selective fluorescent probes to the study of these two important bioinorganic analytes. PMID:21675918

Free metal ion depletion by "Good's" buffers. III. N-(2-acetamido)iminodiacetic acid, 2:1 complexes with zinc(II), cobalt(II), nickel(II), and copper(II); amide deprotonation by Zn(II), Co(II), and Cu(II).

PubMed

Lance, E A; Rhodes, C W; Nakon, R

1983-09-01

Potentiometric, visible, infrared, electron spin, and nuclear magnetic resonance studies of the complexation of N-(2-acetamido)iminodiacetic acid (H2ADA) by Ca(II), Mg(II), Mn(II), Zn(II), Co(II), Ni(II), and Cu(II) are reported. Ca(II) and Mg(II) were found not to form 2:1 ADA2- to M(II) complexes, while Mn(II), Cu(II), Ni(II), Zn(II), and Co(II) did form 2:1 metal chelates at or below physiological pH values. Co(II) and Zn(II), but not Cu(II), were found to induce stepwise deprotonation of the amide groups to form [M(H-1ADA)4-(2)]. Formation (affinity) constants for the various metal complexes are reported, and the probable structures of the various metal chelates in solution are discussed on the basis of various spectral data.

Recognition of thymine in DNA bulges by a Zn(II) macrocyclic complex.

PubMed

del Mundo, Imee Marie A; Fountain, Matthew A; Morrow, Janet R

2011-08-14

A Zn(II) macrocyclic complex with appended quinoline is a bifunctional recognition agent that uses both the Zn(II) center and the pendent aromatic group to bind to thymine in bulges with good selectivity over DNA containing G, C or A bulges. Spectroscopic studies show that the stem containing the bulge stays largely intact in a DNA hairpin with the Zn(II) complex bound to the thymine bulge. This journal is © The Royal Society of Chemistry 2011

A High Affinity Red Fluorescence and Colorimetric Probe for Amyloid β Aggregates

NASA Astrophysics Data System (ADS)

Rajasekhar, K.; Narayanaswamy, Nagarjun; Murugan, N. Arul; Kuang, Guanglin; Ågren, Hans; Govindaraju, T.

2016-04-01

A major challenge in the Alzheimer’s disease (AD) is its timely diagnosis. Amyloid β (Aβ) aggregates have been proposed as the most viable biomarker for the diagnosis of AD. Here, we demonstrate hemicyanine-based benzothiazole-coumarin (TC) as a potential probe for the detection of highly toxic Aβ42 aggregates through switch-on, enhanced (~30 fold) red fluorescence (Emax = 654 nm) and characteristic colorimetric (light red to purple) optical outputs. Interestingly, TC exhibits selectivity towards Aβ42 fibrils compared to other abnormal protein aggregates. TC probe show nanomolar binding affinity (Ka = 1.72 × 107 M-1) towards Aβ42 aggregates and also displace ThT bound to Aβ42 fibrils due to its high binding affinity. The Aβ42 fibril-specific red-shift in the absorption spectra of TC responsible for the observed colorimetric optical output has been attributed to micro-environment change around the probe from hydrophilic-like to hydrophobic-like nature. The binding site, binding energy and changes in optical properties observed for TC upon interaction with Aβ42 fibrils have been further validated by molecular docking and time dependent density functional theory studies.

Synthesis, characterization, crystal structure, DNA- and HSA-binding studies of a dinuclear Schiff base Zn(II) complex derived from 2-hydroxynaphtaldehyde and 2-picolylamine

NASA Astrophysics Data System (ADS)

Kazemi, Zahra; Rudbari, Hadi Amiri; Mirkhani, Valiollah; Sahihi, Mehdi; Moghadam, Majid; Tangestaninejad, Sharam; Mohammadpoor-Baltork, Iraj

2015-09-01

A tridentate Schiff base ligand NNO donor (HL: 1-((E)-((pyridin-2-yl)methylimino)methyl)naphthalen-2-ol was synthesized from condensation of 2-hydroxynaphtaldehyde and 2-picolylamine. Zinc complex, Zn2L2(NO3)2, was prepared from reaction of Zn(NO3)2 and HL at ambient temperature. The ligand and complex were characterized by FT-IR, 1H NMR, 13C NMR and elemental analysis (CHN). Furthermore, the structure of dinuclear Zn(II) complex was determined by single crystal X-ray analysis. The complex, Zn2L2(NO3)2, is centrosymmetric dimer in which deprotonated phenolates bridge the two Zn(II) atoms and link the two halves of the dimer. In the structure, Zinc(II) ions have a highly distorted six-coordinate structure bonded to two oxygen atoms from a bidentate nitrate group, the pyridine nitrogen, an amine nitrogen and phenolate oxygens. The interaction of dinuclear Zn(II) complex with fish sperm DNA (FS-DNA) and HSA was investigated under physiological conditions using fluorescence quenching, UV-Vis spectroscopy, molecular dynamics simulation and molecular docking methods. The estimated binding constants for the DNA-complex and HSA-complex were (3.60 ± 0.18) × 104 M-1 and (1.35 ± 0.24) × 104 M-1, respectively. The distance between dinuclear Zn(II) complex and HSA was obtained based on the Förster's theory of non-radiative energy transfer. Molecular docking studies revealed the binding of dinuclear Zn(II) complex to the major groove of FS-DNA and IIA site of protein by formation of hydrogen bond, π-cation and hydrophobic interactions.

Affinity fluorescence-labeled peptides for the early detection of cancer in Barrett's esophagus

NASA Astrophysics Data System (ADS)

Li, Meng; Lu, Shaoying; Piraka, Cyrus; Appelman, Henry; Kwon, Rich; Soetikno, Roy; Kaltenbach, Tonya; Wang, Thomas D.

2009-02-01

Fluorescence-labeled peptides that affinity bind to neoplastic mucsosa are promising for use as a specific contrast agent in the detection of pre-malignant tissue in the esophagus. This method is can be used to identify expression of biological markers associated with dysplasia on endoscopic imaging as a guide for biopsy and represents a novel method for the early detection and prevention of cancer. We demonstrate the use of phage display to select affinity peptides and identify the sequence "ASYNYDA" that binds with high target-to-background ratio to dysplastic esophageal mucosa compared to that of intestinal metaplasia. Validation of preferential binding is demonstrated for neoplasia in the setting of Barrett's esophagus. An optimal tradeoff between sensitivity and specificity of 82% and 85% was found at the relative threshold of 0.60 with a target-to-background ratio of 1.81 and an area under the ROC curve of 0.87. Peptides are a novel class of ligand for targeted detection of pre-malignant mucosa for purposes of screening and surveillance.

Intercalation of a Zn(II) complex containing ciprofloxacin drug between DNA base pairs.

PubMed

Shahabadi, Nahid; Asadian, Ali Ashraf; Mahdavi, Mryam

2017-11-02

In this study, an attempt has been made to study the interaction of a Zn(II) complex containing an antibiotic drug, ciprofloxacin, with calf thymus DNA using spectroscopic methods. It was found that Zn(II) complex could bind with DNA via intercalation mode as evidenced by: hyperchromism in UV-Vis spectrum; these spectral characteristics suggest that the Zn(II) complex interacts with DNA most likely through a mode that involves a stacking interaction between the aromatic chromophore and the base pairs of DNA. DNA binding constant (K b = 1.4 × 10 4 M -1 ) from spectrophotometric studies of the interaction of Zn(II) complex with DNA is comparable to those of some DNA intercalative polypyridyl Ru(II) complexes 1.0 -4.8 × 10 4 M -1 . CD study showed stabilization of the right-handed B form of DNA in the presence of Zn(II) complex as observed for the classical intercalator methylene blue. Thermodynamic parameters (ΔH < 0 and ΔS < 0) indicated that hydrogen bond and Van der Waals play main roles in this binding prose. Competitive fluorimetric studies with methylene blue (MB) dye have shown that Zn(II) complex exhibits the ability of this complex to displace with DNA-MB, indicating that it binds to DNA in strong competition with MB for the intercalation.

Fluorescence and computational studies of thymidine phosphorylase affinity toward lipidated 5-FU derivatives

NASA Astrophysics Data System (ADS)

Lettieri, R.; D'Abramo, M.; Stella, L.; La Bella, A.; Leonelli, F.; Giansanti, L.; Venanzi, M.; Gatto, E.

2018-04-01

Thymidine phosphorylase (TP) is an enzyme that is up-regulated in a wide variety of solid tumors, including breast and colorectal cancers. It is involved in tumor growth and metastasis, for this reason it is one of the key enzyme to be inhibited, in an attempt to prevent tumor proliferation. However, it also plays an active role in cancer treatment, through its contribution in the conversion of the anti-cancer drug 5-fluorouracil (5-FU) to an irreversible inhibitor of thymidylate synthase (TS), responsible of the inhibition of the DNA synthesis. In this work, the intrinsic TP fluorescence has been investigated for the first time and exploited to study TP binding affinity for the unsubstituted 5-FU and for two 5-FU derivatives, designed to expose this molecule on liposomal membranes. These molecules were obtained by functionalizing the nitrogen atom with a chain consisting of six (1) or seven (2) units of glycol, linked to an alkyl moiety of 12 carbon atoms. Derivatives (1) and (2) exhibited an affinity for TP in the micromolar range, 10 times higher than the parent compound, irrespective of the length of the polyoxyethylenic spacer. This high affinity was maintained also when the compounds were anchored in liposomal membranes. Experimental results were supported by molecular dynamics simulations and docking calculations, supporting a feasible application of the designed supramolecular lipid structure in selective targeting of TP, to be potentially used as a drug delivery system or sensor device.

Quaternized Zn(II) phthalocyanines for photodynamic strategy against resistant periodontal bacteria.

PubMed

Kussovski, Vesselin; Mantareva, Vanya; Durmuş, Mahmut; Angelov, Ivan

2018-04-25

Photodynamic inactivation (PDI) has been featured as an effective strategy in the treatment of acute drug-resistant infections. The efficiency of PDI was evaluated against three periodontal pathogenic bacteria that were tested as drug-resistant strains. In vitro studies were performed with four water-soluble cationic Zn(II) phthalocyanines (ZnPc1-4) and irradiation of a specific light source (light-emitting diode, 665 nm) with three doses (15, 36 and 60 J/cm2). The well detectable fluorescence of ZnPcs allowed the cellular imaging, which suggested relatively high uptakes of ZnPcs into bacterial species. Complete photoinactivation was achieved with all studied ZnPc1-4 for Enterococcus faecalis (E. faecalis) at a light dose of 15 J/cm2. The photodynamic response was high for Prevotella intermedia (P. intermedia) after the application of 6 μM of ZnPc1 and a light dose of 36 J/cm2 and for 6 μM of ZnPc2 at 60 J/cm2. P. intermedia was inactivated with ZnPc3 (4 log) and ZnPc4 (2 log) with irradiation at an optimal dose of 60 J/cm2. Similar photoinactivation results (2 log) were achieved for Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) treated with 6 μM ZnPc1 and ZnPc2 at a light dose of 60 J/cm2. The study suggested that PDI with quaternized Zn(II) phthalocyanines and specific light irradiation appears to be a very useful antimicrobial strategy for effective inactivation of drug-resistant periodontal pathogens.

Intracellular Zn(II) Intoxication Leads to Dysregulation of the PerR Regulon Resulting in Heme Toxicity in Bacillus subtilis

PubMed Central

2016-01-01

Transition metal ions (Zn(II), Cu(II)/(I), Fe(III)/(II), Mn(II)) are essential for life and participate in a wide range of biological functions. Cellular Zn(II) levels must be high enough to ensure that it can perform its essential roles. Yet, since Zn(II) binds to ligands with high avidity, excess Zn(II) can lead to protein mismetallation. The major targets of mismetallation, and the underlying causes of Zn(II) intoxication, are not well understood. Here, we use a forward genetic selection to identify targets of Zn(II) toxicity. In wild-type cells, in which Zn(II) efflux prevents intoxication of the cytoplasm, extracellular Zn(II) inhibits the electron transport chain due to the inactivation of the major aerobic cytochrome oxidase. This toxicity can be ameliorated by depression of an alternate oxidase or by mutations that restrict access of Zn(II) to the cell surface. Conversely, efflux deficient cells are sensitive to low levels of Zn(II) that do not inhibit the respiratory chain. Under these conditions, intracellular Zn(II) accumulates and leads to heme toxicity. Heme accumulation results from dysregulation of the regulon controlled by PerR, a metal-dependent repressor of peroxide stress genes. When metallated with Fe(II) or Mn(II), PerR represses both heme biosynthesis (hemAXCDBL operon) and the abundant heme protein catalase (katA). Metallation of PerR with Zn(II) disrupts this coordination, resulting in depression of heme biosynthesis but continued repression of catalase. Our results support a model in which excess heme partitions to the membrane and undergoes redox cycling catalyzed by reduced menaquinone thereby resulting in oxidative stress. PMID:27935957

[Zn(phen)(O,N,O)(H2O)] and [Zn(phen)(O,N)(H2O)] with O,N,O is 2,6-dipicolinate and N,O is L-threoninate: synthesis, characterization, and biomedical properties.

PubMed

Chin, Lee-Fang; Kong, Siew-Ming; Seng, Hoi-Ling; Tiong, Yee-Lian; Neo, Kian-Eang; Maah, Mohd Jamil; Khoo, Alan Soo-Beng; Ahmad, Munirah; Hor, Tzi-Sum Andy; Lee, Hong-Boon; San, Swee-Lan; Chye, Soi-Moi; Ng, Chew-Hee

2012-10-01

Two ternary Zn(II) complexes, with 1,10-phenanthroline (phen) as the main ligand and a carboxylate-containing ligand [dipicolinate (dipico) or L-threoninate (L-Thr)] as the subsidiary ligand, were prepared and characterized by elemental analysis, Fourier transform IR, UV, and fluorescence spectroscopy, X-ray diffraction, molar conductivity, and electrospray ionization mass spectrometry. X-ray structure analysis shows that both [Zn(phen)(dipico)(H(2)O)]·H(2)O (1) and [Zn(phen)(L-Thr)(H(2)O)Cl]·2H(2)O (2) have octahedral geometry about the Zn(II) atom. Both complexes can inhibit topoisomerase I, and have better anticancer activity than cisplatin against nasopharyngeal cancer cell lines, HK1 and HONE-1, with concentrations causing 50 % inhibition of cell proliferation (IC(50)) in the low micromolar range. Complex 2 has the highest therapeutic index for HK1. Both Zn(II) complexes can induce cell death by apoptosis. Changing the subsidiary ligand in the Zn(II) complexes affects the UV-fluorescence spectral properties of the coordinated phen ligand, the binding affinity for some DNA sequences, nucleobase sequence-selective binding, the phase at which cell cycle progression was arrested for treated cancer cells, and their therapeutic index.

Affinity capillary electrophoresis and fluorescence spectroscopy for studying enantioselective interactions between omeprazole enantiomer and human serum albumin.

PubMed

Xu, Yujing; Hong, Tingting; Chen, Xueping; Ji, Yibing

2017-05-01

Baseline separation of omeprazole (OME) enantiomers was achieved by affinity capillary electrophoresis (ACE), using human serum albumin (HSA) as the chiral selector. The influence of several experimental variables such as HSA concentration, the type and content of organic modifiers, applied voltage and running buffer concentration on the separation was evaluated. The binding of esomeprazole (S-omeprazole, S-OME) and its R-enantiomer (R-omeprazole, R-OME) to HSA under simulated physiological conditions was studied by ACE and fluorescence spectroscopy which was considered as a reference method. ACE studies demonstrated that the binding constants of the two enantiomers and HSA were 3.18 × 10 3 M -1 and 5.36 × 10 3 M -1 , respectively. The binding properties including the fluorescence quenching mechanisms, binding constants, binding sites and the number of binding sites were obtained by fluorescence spectroscopy. Though the ACE method could not get enough data when compared with the fluorescence spectrum method, the separation and binding studies of chiral drugs could be achieved simultaneously via this method. This study is of great significance for the investigation and clinical application of chiral drugs. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Determining the ice-binding planes of antifreeze proteins by fluorescence-based ice plane affinity.

PubMed

Basu, Koli; Garnham, Christopher P; Nishimiya, Yoshiyuki; Tsuda, Sakae; Braslavsky, Ido; Davies, Peter

2014-01-15

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms.

Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity

PubMed Central

Basu, Koli; Garnham, Christopher P.; Nishimiya, Yoshiyuki; Tsuda, Sakae; Braslavsky, Ido; Davies, Peter

2014-01-01

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms. PMID:24457629

A Series of Zn(II) Terpyridine-Based Nitrate Complexes as Two-Photon Fluorescent Probe for Identifying Apoptotic and Living Cells via Subcellular Immigration.

PubMed

Liu, Dandan; Zhang, Mingzhu; Du, Wei; Hu, Lei; Li, Fei; Tian, Xiaohe; Wang, Aidong; Zhang, Qiong; Zhang, Zhongping; Wu, Jieying; Tian, Yupeng

2018-06-19

Two-photon active probe to label apoptotic cells plays a significant role in biological systems. However, discrimination of live/apoptotic cells at subcellular level under microscopy remains unachieved. Here, three novel Zn(II) terpyridine-based nitrate complexes (C1-C3) containing different pull/push units were designed. The structures of the ligands and their corresponding Zn(II) complexes were confirmed by single-crystal X-ray diffraction analysis. On the basis of the comprehensive comparison, C3 had a suitable two-photon absorption cross section in the near-infrared wavelength and good biocompatibility. Under two-photon confocal microscopy and transmission electron microscopy, it is found that C3 could target mitochondria in living cells but immigrate into the nucleolus during the apoptotic process. This dual-functional probe (C3) not only offers a valuable image tool but also acts as an indicator for cell mortality at subcellular level in a real-time manner.

Fluorogen-Activating-Proteins as Universal Affinity Biosensors for Immunodetection

PubMed Central

Gallo, Eugenio; Vasilev, Kalin V.; Jarvik, Jonathan

2014-01-01

Fluorogen-activating-proteins (FAPs) are a novel platform of fluorescence biosensors utilized for protein discovery. The technology currently demands molecular manipulation methods that limit its application and adaptability. Here, we highlight an alternative approach based on universal affinity reagents for protein detection. The affinity reagents were engineered as bi-partite fusion proteins, where the specificity moiety is derived from IgG-binding proteins –Protein-A or Protein-G – and the signaling element is a FAP. In this manner, primary antibodies provide the antigenic selectivity against a desired protein in biological samples, while FAP affinity reagents target the constant region (Fc) of antibodies and provide the biosensor component of detection. Fluorescence results using various techniques indicate minimal background and high target specificity for exogenous and endogenous proteins in mammalian cells. Additionally, FAP-based affinity reagents provide enhanced properties of detection previously absent using conventional affinity systems. Distinct features explored in this report include: (1) unfixed signal wavelengths (excitation and emission) determined by the particular fluorogen chosen, (2) real-time user controlled fluorescence on-set and off-set, (3) signal wavelength substitution while performing live analysis, and (4) enhanced resistance to photobleaching. PMID:24122476

Synthesis Characterization and DNA Interaction Studies of a New Zn(II) Complex Containing Different Dinitrogen Aromatic Ligands

PubMed Central

Shahabadi, Nahid; Mohammadi, Somaye

2012-01-01

A mononuclear complex of Zn(II), [Zn(DIP)2 (DMP)] (NO3)2 ·2H2O in which DIP is 4,7-diphenyl-1,10-phenanthroline and DMP is 4,4′-dimethyl-2,2′-bipyridine has been prepared and characterized by 1HNMR spectroscopy, FT-IR, UV-Vis and elemental analysis techniques. DNA-binding properties of the complex were studied using UV-vis spectra, circular dichroism (CD) spectra, fluorescence, cyclic voltammetry (CV), and viscosity measurements. The results indicate that this zinc(II) complex can intercalate into the stacked base pairs of DNA and compete with the strong intercalator ethidium bromide for the intercalative binding sites. PMID:22956919

Ap4A is not an efficient Zn(II) binding agent. A concerted potentiometric, calorimetric and NMR study.

PubMed

Wszelaka-Rylik, Małgorzata; Witkiewicz-Kucharczyk, Aleksandra; Wójcik, Jacek; Bal, Wojciech

2007-05-01

Diadenosine 5',5''-P(1)P(4) tetraphosphate (Ap(4)A) has been considered as an intracellular partner for Zn(II). We applied potentiometry, ITC and NMR to study protonation equilibria of Ap(4)A and Zn(II) complexation by this dinucleotide. The values of binding constants obtained by these three techniques under various experimental conditions coherently demonstrated that Ap(4)A binds Zn(II) weakly, with an apparent binding constant of ca. 10(4) at neutral pH. Such a low stability of Zn(II) complexes with Ap(4)A excludes a possibility for interactions between these two agents in vivo.

Generating a Metal-responsive Transcriptional Regulator to Test What Confers Metal Sensing in Cells*

PubMed Central

Osman, Deenah; Piergentili, Cecilia; Chen, Junjun; Chakrabarti, Buddhapriya; Foster, Andrew W.; Lurie-Luke, Elena; Huggins, Thomas G.; Robinson, Nigel J.

2015-01-01

FrmR from Salmonella enterica serovar typhimurium (a CsoR/RcnR-like transcriptional de-repressor) is shown to repress the frmRA operator-promoter, and repression is alleviated by formaldehyde but not manganese, iron, cobalt, nickel, copper, or Zn(II) within cells. In contrast, repression by a mutant FrmRE64H (which gains an RcnR metal ligand) is alleviated by cobalt and Zn(II). Unexpectedly, FrmR was found to already bind Co(II), Zn(II), and Cu(I), and moreover metals, as well as formaldehyde, trigger an allosteric response that weakens DNA affinity. However, the sensory metal sites of the cells' endogenous metal sensors (RcnR, ZntR, Zur, and CueR) are all tighter than FrmR for their cognate metals. Furthermore, the endogenous metal sensors are shown to out-compete FrmR. The metal-sensing FrmRE64H mutant has tighter metal affinities than FrmR by approximately 1 order of magnitude. Gain of cobalt sensing by FrmRE64H remains enigmatic because the cobalt affinity of FrmRE64H is substantially weaker than that of the endogenous cobalt sensor. Cobalt sensing requires glutathione, which may assist cobalt access, conferring a kinetic advantage. For Zn(II), the metal affinity of FrmRE64H approaches the metal affinities of cognate Zn(II) sensors. Counter-intuitively, the allosteric coupling free energy for Zn(II) is smaller in metal-sensing FrmRE64H compared with nonsensing FrmR. By determining the copies of FrmR and FrmRE64H tetramers per cell, then estimating promoter occupancy as a function of intracellular Zn(II) concentration, we show how a modest tightening of Zn(II) affinity, plus weakened DNA affinity of the apoprotein, conspires to make the relative properties of FrmRE64H (compared with ZntR and Zur) sufficient to sense Zn(II) inside cells. PMID:26109070

Fusion of a Novel Genetically Engineered Chitosan Affinity Protein and Green Fluorescent Protein for Specific Detection of Chitosan In Vitro and In Situ

PubMed Central

Nampally, Malathi; Moerschbacher, Bruno Maria

2012-01-01

Chitin is the second most abundant polysaccharide, present, e.g., in insect and arthropod exoskeletons and fungal cell walls. In some species or under specific conditions, chitin appears to be enzymatically de-N-acetylated to chitosan—e.g., when pathogenic fungi invade their host tissues. Here, the deacetylation of chitin is assumed to represent a pathogenicity mechanism protecting the fungus from the host's chitin-driven immune response. While highly specific chitin binding lectins are well known and easily available, this is not the case for chitosan-specific probes. This is partly due to the poor antigenicity of chitosan so that producing high-affinity, specific antibodies is difficult. Also, lectins with specificity to chitosan have been described but are not commercially available, and our attempts to reproduce the findings were not successful. We have, therefore, generated a fusion protein between a chitosanase inactivated by site-directed mutagenesis, the green fluorescent protein (GFP), and StrepII, as well as His6 tags for purification and detection. The recombinant chitosan affinity protein (CAP) expressed in Escherichia coli was shown to specifically bind to chitosan, but not to chitin, and the affinity increased with decreasing degree of acetylation. In vitro, CAP detection was possible either based on GFP fluorescence or using Strep-Tactin conjugates or anti-His5 antibodies. CAP fluorescence microscopy revealed binding to the chitosan exposing endophytic infection structures of the wheat stem rust fungus, but not the chitin exposing ectophytic infection structures, verifying its suitability for in situ chitosan staining. PMID:22367086

Electrochemical immobilization of Fluorescent labelled probe molecules on a FTO surface for affinity detection based on photo-excited current

NASA Astrophysics Data System (ADS)

Haruyama, Tetsuya; Wakabayashi, Ryo; Cho, Takeshi; Matsuyama, Sho-taro

2011-10-01

Photo-excited current can be generated at a molecular interface between a photo-excited molecules and a semi-conductive material in appropriate condition. The system has been recognized for promoting photo-energy devices such as an organic dye sensitized solar-cell. The photo-current generated reactions are totally dependent on the interfacial energy reactions, which are in a highly fluctuated interfacial environment. The authors investigated the photo-excited current reaction to develop a smart affinity detection method. However, in order to perform both an affinity reaction and a photo-excited current reaction at a molecular interface, ordered fabrications of the functional (affinity, photo-excitation, etc.) molecules layer on a semi-conductive surface is required. In the present research, we would like to present the fabrication and functional performance of photo-excited current-based affinity assay device and its application for detection of endocrine disrupting chemicals. On the FTO surface, fluorescent pigment labelled affinity peptide was immobilized through the EC tag (electrochemical-tag) method. The modified FTO produced a current when it was irradiated with diode laser light. However, the photo current decreased drastically when estrogen (ES) coexisted in the reaction solution. In this case, immobilized affinity probe molecules formed a complex with ES and estrogen receptor (ER). The result strongly suggests that the photo-excited current transduction between probe molecule-labelled cyanine pigment and the FTO surface was partly inhibited by a complex that formed at the affinity oligo-peptide region in a probe molecule on the FTO electrode. The bound bulky complex may act as an impediment to perform smooth transduction of photo-excited current in the molecular interface. The present system is new type of photo-reaction-based analysis. This system can be used to perform simple high-sensitive homogeneous assays.

Adsorption of Pb(II), Cu(II), Cd(II), Zn(II), Ni(II), Fe(II), and As(V) on bacterially produced metal sulfides.

PubMed

Jong, Tony; Parry, David L

2004-07-01

The adsorption of Pb(II), Cu(II), Cd(II), Zn(II), Ni(II), Fe(II) and As(V) onto bacterially produced metal sulfide (BPMS) material was investigated using a batch equilibrium method. It was found that the sulfide material had adsorptive properties comparable with those of other adsorbents with respect to the specific uptake of a range of metals and, the levels to which dissolved metal concentrations in solution can be reduced. The percentage of adsorption increased with increasing pH and adsorbent dose, but decreased with increasing initial dissolved metal concentration. The pH of the solution was the most important parameter controlling adsorption of Cd(II), Cu(II), Fe(II), Ni(II), Pb(II), Zn(II), and As(V) by BPMS. The adsorption data were successfully modeled using the Langmuir adsorption isotherm. Desorption experiments showed that the reversibility of adsorption was low, suggesting high-affinity adsorption governed by chemisorption. The mechanism of adsorption for the divalent metals was thought to be the formation of strong, inner-sphere complexes involving surface hydroxyl groups. However, the mechanism for the adsorption of As(V) by BPMS appears to be distinct from that of surface hydroxyl exchange. These results have important implications to the management of metal sulfide sludge produced by bacterial sulfate reduction.

A flexible 1,8-naphthyridyl derivative and its Zn(II) complexes: synthesis, structures, spectroscopic properties and recognition of Cd(II).

PubMed

Zhang, Hui-Miao; Fu, Wen-Fu; Gan, Xin; Xu, Yan-Qing; Wang, Jun; Xu, Quan-Qing; Chi, Shao-Ming

2008-12-21

A flexible ligand bis(7-methyl-1,8-naphthyridine-2-ylamino)methane (), having kappa(4)-chelating and kappa(2)-bridging modes, and its intriguing structural complexes of Zn(II) with mu-OH, kappa(1)-OAc, mu-kappa(1)-OAc and mu-kappa(2)-OAc ligands, [Zn(2)()(2)(OH)](ClO(4))(3) (), [Zn(4)()(2)(OAc)(6)(OH)(2)].CH(2)Cl(2) (.CH(2)Cl(2)) and [Zn(5)()(2)(OAc)(10)](n).4nH(2)O (.4H(2)O) were synthesized and their structures were determined by X-ray crystallography. These compounds exhibited intense blue fluorescent emissions with a lambda(max) in the range of 380-410 nm in CH(2)Cl(2), CH(3)CN and CH(3)OH solutions, and solid-state emissions centered at 416, 463, 490 and 451 nm were observed for the compounds , , and at room temperature, respectively. The investigated fluorescence properties of associated with various metal ions showed that the fluorescence enhancement of with Cd(II) was more sensitive than with other interfering cations.

Synthetic hydroxyapatites doped with Zn(II) studied by X-ray diffraction, infrared, Raman and thermal analysis

NASA Astrophysics Data System (ADS)

Guerra-López, José R.; Echeverría, Gustavo A.; Güida, Jorge A.; Viña, Raúl; Punte, Graciela

2015-06-01

Calcium hydroxyapatite (CaHap) formation when different amounts of Zn(II) are present in the mother solution has been investigated by atomic absorption, infrared and Raman spectroscopies, X-ray diffraction and thermal analysis (DTA and TG). The studied samples have been synthesized at T=95 °C and pH 9 in air. The analysis of the results have shown that the pure CaHap sample crystallizes in the monoclinic form P21/b. Concentrations up to 20% of Zn(II) in the mother solution, equivalent to smaller concentrations in solid (up to 9.1% in wt), favor the formation of the hexagonal apatite, P63/m, while Zn(II) concentrations higher than 20% in solution help an amorphous phase development where vibrational spectra indicated coexistence of two phases: an apatite and ZnNH4PO4·H2O. Infrared data of thermal treated samples endorse that HPO42- ion had not been incorporated in Zn(II) doped samples during the synthesis process. Present results also allow to conclude that Zn(II) cation exhibits a preference to occupy the Ca2 site of the apatite structure and induces water adsorption and a small quantity of CO32- cation incorporation, leading to formation of a less crystalline Ca deficient apatite.

Synthesis, spectral and third-order nonlinear optical properties of terpyridine Zn(II) complexes based on carbazole derivative with polyether group

NASA Astrophysics Data System (ADS)

Kong, Ming; Liu, Yanqiu; Wang, Hui; Luo, Junshan; Li, Dandan; Zhang, Shengyi; Li, Shengli; Wu, Jieying; Tian, Yupeng

2015-01-01

Four novel Zn(II) terpyridine complexes (ZnLCl2, ZnLBr2, ZnLI2, ZnL(SCN)2) based on carbazole derivative group were designed, synthesized and fully characterized. Their photophysical properties including absorption and one-photon excited fluorescence, two-photon absorption (TPA) and optical power limiting (OPL) were further investigated systematically and interpreted on the basis of theoretical calculations (TD-DFT). The influences of different solvents on the absorption and One-Photon Excited Fluorescence (OPEF) spectral behavior, quantum yields and the lifetime of the chromophores have been investigated in detail. The third-order nonlinear optical (NLO) properties were investigated by open/closed aperture Z-scan measurements using femtosecond pulse laser in the range from 680 to 1080 nm. These results revealed that ZnLCl2 and ZnLBr2 exhibited strong two-photon absorption and ZnLCl2 showed superior optical power limiting property.

Zn(II) and Hg(II) binding to a designed peptide that accommodates different coordination geometries.

PubMed

Szunyogh, Dániel; Gyurcsik, Béla; Larsen, Flemming H; Stachura, Monika; Thulstrup, Peter W; Hemmingsen, Lars; Jancsó, Attila

2015-07-28

Designed metal ion binding peptides offer a variety of applications in both basic science as model systems of more complex metalloproteins, and in biotechnology, e.g. in bioremediation of toxic metal ions, biomining or as artificial enzymes. In this work a peptide (HS: Ac-SCHGDQGSDCSI-NH2) has been specifically designed for binding of both Zn(II) and Hg(II), i.e. metal ions with different preferences in terms of coordination number, coordination geometry, and to some extent ligand composition. It is demonstrated that HS accommodates both metal ions, and the first coordination sphere, metal ion exchange between peptides, and speciation are characterized as a function of pH using UV-absorption-, synchrotron radiation CD-, (1)H-NMR-, and PAC-spectroscopy as well as potentiometry. Hg(II) binds to the peptide with very high affinity in a {HgS2} coordination geometry, bringing together the two cysteinates close to each end of the peptide in a loop structure. Despite the high affinity, Hg(II) is kinetically labile, exchanging between peptides on the subsecond timescale, as indicated by line broadening in (1)H-NMR. The Zn(II)-HS system displays more complex speciation, involving monomeric species with coordinating cysteinates, histidine, and a solvent water molecule, as well as HS-Zn(II)-HS complexes. In summary, the HS peptide displays conformational flexibility, contains many typical metal ion binding groups, and is able to accommodate metal ions with different structural and ligand preferences with high affinity. As such, the HS peptide may be a scaffold offering binding of a variety of metal ions, and potentially serve for metal ion sequestration in biotechnological applications.

Facile synthesis of 2D Zn(II) coordination polymer and its crystal structure, selective removal of methylene blue and molecular simulations

NASA Astrophysics Data System (ADS)

Sezer, Güneş Günay; Yeşilel, Okan Zafer; Şahin, Onur; Arslanoğlu, Hasan; Erucar, İlknur

2017-09-01

A new coordination polymer {[Zn(μ3-ppda)(H2O)(μ-bpa)Zn(μ-ppda)(μ-bpa)]·4H2O}n (1) (ppda = 1,4-phenylenediacetate, bpa = 1,2-bis(4-pyridyl)ethane) has been synthesized by microwave-assisted reaction and characterized by elemental analysis, IR spectroscopy, single-crystal and powder X-ray diffractions. The asymmetric unit of 1 consists of two Zn(II) ions, two bpa ligands, two ppda ligands, one coordinated and four non-coordinated water molecules. In 1, ppda2- anions are linked the adjacent Zn(II) centers to generate 1D double-stranded chains. These chains are connected into 2D sheets by the bridging bpa ligands. Atomically detailed modeling was performed to compute single and binary component adsorption isotherms of H2, CO2, CH4 and N2 in complex 1. Results showed that 1 exhibits a high adsorption selectivity towards CO2 due to its high affinity for CO2. Results of this study will be helpful to guide the microwave-assisted reaction of coordination polymers to design promising adsorbents for gas storage and gas separation applications. The luminescent property of 1 and the selective removal of dyes in 1 have been also discussed. Results showed that 1 can be a potential candidate for luminescence applications and can selectively adsorb methylene blue (MB) dye molecules.

A human pilot study of the fluorescence affinity sensor for continuous glucose monitoring in diabetes.

PubMed

Dutt-Ballerstadt, Ralph; Evans, Colton; Pillai, Arun P; Orzeck, Eric; Drabek, Rafal; Gowda, Ashok; McNichols, Roger

2012-03-01

We report results of a pilot clinical study of a subcutaneous fluorescence affinity sensor (FAS) for continuous glucose monitoring conducted in people with type 1 and type 2 diabetes. The device was assessed based on performance, safety, and comfort level under acute conditions (4 h). A second-generation FAS (BioTex Inc., Houston, TX) was subcutaneously implanted in the abdomens of 12 people with diabetes, and its acute performance to excursions in blood glucose was monitored over 4 h. After 30-60 min the subjects, who all had fasting blood glucose levels of less than 200 mg/dl, received a glucose bolus of 75 g/liter dextrose by oral administration. Capillary blood glucose samples were obtained from the finger tip. The FAS data were retrospectively evaluated by linear least squares regression analysis and by the Clarke error grid method. Comfort levels during insertion, operation, and sensor removal were scored by the subjects using an analog pain scale. After retrospective calibration of 17 sensors implanted in 12 subjects, error grid analysis showed 97% of the paired values in zones A and B and 1.5% in zones C and D, respectively. The mean absolute relative error between sensor signal and capillary blood glucose was 13% [±15% standard deviation (SD), 100-350 mg/dl] with an average correlation coefficient of 0.84 (±0.24 SD). The actual average "warm-up" time for the FAS readings, at which highest correlation with glucose readings was determined, was 65 (±32 SD) min. Mean time lag was 4 (±5 SD) min during the initial operational hours. Pain levels during insertion and operation were modest. The in vivo performance of the FAS demonstrates feasibility of the fluorescence affinity technology to determine blood glucose excursions accurately and safely under acute dynamic conditions in humans with type 1 and type 2 diabetes. Specific engineering challenges to sensor and instrumentation robustness remain. Further studies will be required to validate its promising

Time-dependent Translational Response of E. coli to Excess Zn(II)

PubMed Central

Easton, J. Allen; Thompson, Peter; Crowder, Michael W.

2006-01-01

Zinc homeostasis is not well understood beyond methods of import and export. In order to better understand zinc homeostasis in Escherichia coli by identifying Zn(ii)-responsive proteins, a proteomic approach was taken. Through the use of two-dimensional gel electrophoresis, we were able to show that the levels of OmpF, AspC, YcdO, Eno, and CysE increased after 30 min of Zn(ii) stress, while the levels of Tig, TufA, SelA, and LeuC decreased relative to non-stressed controls. After 4 h of Zn(ii) stress, the levels of three proteins (DnaK, YeaU, and Mdh) were found to be up-regulated, while the levels of seven amino acid importers (HisJ, ArgT, LivJ, DppA, OppA, RbsB, and GinH) were found to be decreased. None of these proteins had been reported to be up- or down-regulated in any previously published cDNA microarray experiments. This result raises questions about the validity of cDNA arrays when they are used to make assumptions concerning protein levels within bacterial cells. These data also suggest that time is a factor when characterizing how the E. coli proteome responds to Zn(ii) stress. PMID:17122063

Characterization of sophorolipid biosurfactant produced by Cryptococcus sp. VITGBN2 and its application on Zn(II) removal from electroplating wastewater.

PubMed

Basak, Geetanjali; Das, Nilanjana

2014-11-01

The present study aimed at elucidating the role of biosurfactant produced by yeast for the removal of Zn(II) ions from electroplating wastewater. The yeast species isolated from CETP, Vellore, Tamilnadu was identified as Cryptococcus sp.VITGBN2, based on molecular techniques, and was found to be potent producer of biosurfactant in mineral salt media containing vegetable oil as additional carbon source. Chemical structure of the purified biosurfactant was identified as acidic diacetate sophorolipid through GC-MS analysis. Interaction of Zn(II) ions with biosurfactant was monitored using FT-IR, SEM and EDS analysis. Zn (II) removal at 100 mg l(-1) concentration was 84.8% compared were other synthetic surfactants (Tween 80 and sodium dodecyl sulphate), yeast mediated biosurfactant showed enhanced Zn (II) removal in batch mode. The role of biosurfactant on Zn(II) removal was evaluated in column mode packed with biosurfactant entrapped in sodium alginate beads. At a flow rate of 1 ml min(-1) and bed height of 12 cm, immobilized biosurfactant showed 94.34% Zn(II) removal from electroplating wastewater. The present study confirmed that Zn(II) removal was biosurfactant mediated. This is the first report establishing the involvement of yeast mediated biosurfactant in Zn(II) removal from wastewater.

Adsorption of enrofloxacin in presence of Zn(II) on a calcareous soil.

PubMed

Graouer-Bacart, Mareen; Sayen, Stéphanie; Guillon, Emmanuel

2015-12-01

As a result of their consumption, excretion, disposal and persistence, antibiotics enter the soil environment and may be transported to surface and ground waters. During their transfer through soils, retention processes play a key role in their mobility. Antibiotics often coexist with heavy metals in soils due to agricultural practices and other sources of inputs. In this context, this study deals with the co-adsorption of Zn(II) and enrofloxacin (ENR), a widely-used veterinary antibiotic, on a calcareous soil using batch retention experiments and X-ray Absorption Near Edge Structure (XANES) spectroscopy. To improve our understanding of the interaction of this emerging organic contaminant with metal cations at the water-soil interface, the ternary system containing ENR, Zn(II) and a selected calcareous soil was investigated over a pH range between 7 and 10, at different solid-solution contact times and ENR concentrations. The presence of Zn(II) slightly influenced the retention of the antibiotic, leading to an increase of the adsorbed ENR amounts. The distribution coefficient Kd value increased from 0.66 Lg(-1) for single ENR adsorption to 1.04 Lg(-1) in presence of Zn(II) at a 1/2 ENR/Zn(II) ratio. The combination of adsorption isotherm data, solution speciation diagrams and XANES spectra evidenced a small proportion of Zn(II)-ENR complexes at soil pH leading to the slight increase of ENR adsorption in presence of zinc. These results suggest that it is necessary to consider the interaction between ENR and metal cations when assessing the mobility of ENR in soils. Copyright © 2015 Elsevier Inc. All rights reserved.

Batch and fixed-bed column studies for biosorption of Zn(II) ions onto pongamia oil cake (Pongamia pinnata) from biodiesel oil extraction.

PubMed

Shanmugaprakash, M; Sivakumar, V

2015-12-01

The present work, analyzes the potential of defatted pongamia oil cake (DPOC) for the biosorption of Zn(II) ions from aqueous solutions in the both batch and column mode. Batch experiments were conducted to evaluate the optimal pH, effect of adsorbent dosage, initial Zn(II) ions concentration and contact time. The biosorption equilibrium and kinetics data for Zn(II) ions onto the DPOC were studied in detail, using several models, among all it was found to be that, Freundlich and the second-order model explained the equilibrium data well. The calculated thermodynamic parameters had shown that the biosorption of Zn(II) ions was exothermic and spontaneous in nature. Batch desorption studies showed that the maximum Zn(II) recovery occurred, using 0.1 M EDTA. The Bed Depth Service Time (BDST) and the Thomas model was successfully employed to evaluate the model parameters in the column mode. The results indicated that the DPOC can be applied as an effective and eco-friendly biosorbent for the removal of Zn(II) ions in polluted wastewater. Copyright © 2015 Elsevier Ltd. All rights reserved.

Zn(II)- and Cu(II)-induced non-fibrillar aggregates of amyloid-beta (1-42) peptide are transformed to amyloid fibrils, both spontaneously and under the influence of metal chelators.

PubMed

Tõugu, Vello; Karafin, Ann; Zovo, Kairit; Chung, Roger S; Howells, Claire; West, Adrian K; Palumaa, Peep

2009-09-01

Aggregation of amyloid-beta (Abeta) peptides is a central phenomenon in Alzheimer's disease. Zn(II) and Cu(II) have profound effects on Abeta aggregation; however, their impact on amyloidogenesis is unclear. Here we show that Zn(II) and Cu(II) inhibit Abeta(42) fibrillization and initiate formation of non-fibrillar Abeta(42) aggregates, and that the inhibitory effect of Zn(II) (IC(50) = 1.8 micromol/L) is three times stronger than that of Cu(II). Medium and high-affinity metal chelators including metallothioneins prevented metal-induced Abeta(42) aggregation. Moreover, their addition to preformed aggregates initiated fast Abeta(42) fibrillization. Upon prolonged incubation the metal-induced aggregates also transformed spontaneously into fibrils, that appear to represent the most stable state of Abeta(42). H13A and H14A mutations in Abeta(42) reduced the inhibitory effect of metal ions, whereas an H6A mutation had no significant impact. We suggest that metal binding by H13 and H14 prevents the formation of a cross-beta core structure within region 10-23 of the amyloid fibril. Cu(II)-Abeta(42) aggregates were neurotoxic to neurons in vitro only in the presence of ascorbate, whereas monomers and Zn(II)-Abeta(42) aggregates were non-toxic. Disturbed metal homeostasis in the vicinity of zinc-enriched neurons might pre-dispose formation of metal-induced Abeta aggregates, subsequent fibrillization of which can lead to amyloid formation. The molecular background underlying metal-chelating therapies for Alzheimer's disease is discussed in this light.

Discovery of PF-06928215 as a high affinity inhibitor of cGAS enabled by a novel fluorescence polarization assay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hall, Justin; Brault, Amy; Vincent, Fabien

Cyclic GMP-AMP synthase (cGAS) initiates the innate immune system in response to cytosolic dsDNA. After binding and activation from dsDNA, cGAS uses ATP and GTP to synthesize 2', 3' -cGAMP (cGAMP), a cyclic dinucleotide second messenger with mixed 2'-5' and 3'-5' phosphodiester bonds. Inappropriate stimulation of cGAS has been implicated in autoimmune disease such as systemic lupus erythematosus, thus inhibition of cGAS may be of therapeutic benefit in some diseases; however, the size and polarity of the cGAS active site makes it a challenging target for the development of conventional substrate-competitive inhibitors. We report here the development of a highmore » affinity (K D = 200 nM) inhibitor from a low affinity fragment hit with supporting biochemical and structural data showing these molecules bind to the cGAS active site. We also report a new high throughput cGAS fluorescence polarization (FP)-based assay to enable the rapid identification and optimization of cGAS inhibitors. This FP assay uses Cy5-labelled cGAMP in combination with a novel high affinity monoclonal antibody that specifically recognizes cGAMP with no cross reactivity to cAMP, cGMP, ATP, or GTP. Given its role in the innate immune response, cGAS is a promising therapeutic target for autoinflammatory disease. Our results demonstrate its druggability, provide a high affinity tool compound, and establish a high throughput assay for the identification of next generation cGAS inhibitors.« less

Removal of Zn(II) from electroplating effluent using yeast biofilm formed on gravels: batch and column studies

PubMed Central

2014-01-01

Background Present study deals with the removal of Zn(II) ions from effluent using yeast biofilm formed on gravels. Methods The biofilm forming ability of Candida rugosa and Cryptococcus laurentii was evaluated using XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) reduction assay and monitored by scanning electron microscopy (SEM), and Confocal laser scanning microscopy (CLSM). Copious amount of extracellular polymeric substances (EPS) produced by yeast species was quantified and characterized by Fourier transform infrared spectroscopy (FT-IR). Results Yeast biofilm formed on gravels by C. rugosa and C. laurentii showed 88% and 74.2% removal of Zn(II) ions respectively in batch mode. In column mode, removal of Zn(II) ions from real effluent was found to be 95.29% by C. rugosa biofilm formed on gravels. Conclusion The results of the present study showed that there is a scope to develop a cost effective method for the efficient removal of Zn(II) from effluent using gravels coated with yeast biofilm. PMID:24397917

Structural characterization of 1,8-naphthalimides and in vitro microbiological activity of their Cu(II) and Zn(II) complexes

NASA Astrophysics Data System (ADS)

Grabchev, Ivo; Yordanova, Stanislava; Bosch, Paula; Vasileva-Tonkova, Evgenia; Kukeva, Rositsa; Stoyanov, Stanimir; Stoyanova, Radostina

2017-02-01

Two new 1,8-naphthalimide derivatives (NI1 and NI2) have been synthesized and characterized. The photophysical properties of the new compounds have been investigated in organic solvents of different polarity. It has been shown that both compounds are solvent depended. Cu(II) and Zn(II) complexes of NI2 were obtained and characterized by IR-NMR, fluorescence and EPR spectroscopy. The influence of different metal cations on the fluorescence intensity has been investigated in acetonitrile solution. Antimicrobial composite PLA-metal complexes materials have been obtained for the first time. Microbiological activity of both metal complexes has been investigated in vitro against different Gram-positive and Gram-negative bacteria and two yeasts. The various antimicrobial activities and the minimum inhibitory concentrations (MICs) of both complexes have been determined. The microbiological activity of composite materials PLA-metal complexes in thin polymeric film has also been investigated. The results suggest that the new metal complexes could find application in designing new antimicrobial preparations to control the spread of infections.

Novel fluorescent labelled affinity probes for diadenosine-5',5'''-P1,P4-tetraphosphate (Ap4A)-binding studies.

PubMed

Wright, Michael; Miller, Andrew D

2006-02-15

Tandem synthetic-biosynthetic procedures were used to prepare two novel fluorescent labelled affinity probes for diadenosine-5',5'''-P1,P4-tetraphosphate (Ap4A)-binding studies. These compounds (dial-mant-Ap4A and azido-mant-Ap4A) are shown to clearly distinguish known Ap4A-binding proteins from Escherichia coli (LysU and GroEL) and a variety of other control proteins. Successful labelling of chaperonin GroEL appears to be allosteric with respect to the well-characterized adenosine 5'-triphosphate (ATP)-binding site, suggesting that GroEL possesses a distinct Ap4A-binding site.

Visualizing the kinetic power stroke that drives proton-coupled Zn(II) transport

PubMed Central

Gupta, Sayan; Chai, Jin; Cheng, Jie; D'Mello, Rhijuta; Chance, Mark R.; Fu, Dax

2014-01-01

The proton gradient is a principal energy source for respiration-dependent active transport, but the structural mechanisms of proton-coupled transport processes are poorly understood. YiiP is a proton-coupled zinc transporter found in the cytoplasmic membrane of E. coli, and the transport-site of YiiP receives protons from water molecules that gain access to its hydrophobic environment and transduces the energy of an inward proton gradient to drive Zn(II) efflux1,2. This membrane protein is a well characterized member3-7 of the protein family of cation diffusion facilitators (CDFs) that occurs at all phylogenetic levels8-10. X-ray mediated hydroxyl radical labeling of YiiP and mass spectrometric analysis showed that Zn(II) binding triggered a highly localized, all-or-none change of water accessibility to the transport-site and an adjacent hydrophobic gate. Millisecond time-resolved dynamics revealed a concerted and reciprocal pattern of accessibility changes along a transmembrane helix, suggesting a rigid-body helical reorientation linked to Zn(II) binding that triggers the closing of the hydrophobic gate. The gated water access to the transport-site enables a stationary proton gradient to facilitate the conversion of zinc binding energy to the kinetic power stroke of a vectorial zinc transport. The kinetic details provide energetic insights into a proton-coupled active transport reaction. PMID:25043033

Chelation of Cu(II), Zn(II), and Fe(II) by tannin constituents of selected edible nuts.

PubMed

Karamać, Magdalena

2009-12-22

The tannin fractions isolated from hazelnuts, walnuts and almonds were characterised by colorimetric assays and by an SE-HPLC technique. The complexation of Cu(II) and Zn(II) was determined by the reaction with tetramethylmurexide, whereas for Fe(II), ferrozine was employed. The walnut tannins exhibited a significantly weaker reaction with the vanillin/HCl reagent than hazelnut and almond tannins, but the protein precipitation capacity of the walnut fraction was high. The SE-HPLC chromatogram of the tannin fraction from hazelnuts revealed the presence of oligomers with higher molecular weights compared to that of almonds. Copper ions were most effectively chelated by the constituents of the tannin fractions of hazelnuts, walnuts and almonds. At a 0.2 mg/assay addition level, the walnut tannins complexed almost 100% Cu(II). The Fe(II) complexation capacities of the tannin fractions of walnuts and hazelnuts were weaker in comparison to that of the almond tannin fraction, which at a 2.5 mg/assay addition level, bound Fe(II) by approximately 90%. The capacity to chelate Zn(II) was quite varied for the different nut tannin fractions: almond tannins bound as much as 84% Zn(II), whereas the value for walnut tannins was only 8.7%; and for hazelnut tannins, no Zn(II) chelation took place at the levels tested.

Chelation of Cu(II), Zn(II), and Fe(II) by Tannin Constituents of Selected Edible Nuts

PubMed Central

Karamać, Magdalena

2009-01-01

The tannin fractions isolated from hazelnuts, walnuts and almonds were characterised by colorimetric assays and by an SE-HPLC technique. The complexation of Cu(II) and Zn(II) was determined by the reaction with tetramethylmurexide, whereas for Fe(II), ferrozine was employed. The walnut tannins exhibited a significantly weaker reaction with the vanillin/HCl reagent than hazelnut and almond tannins, but the protein precipitation capacity of the walnut fraction was high. The SE-HPLC chromatogram of the tannin fraction from hazelnuts revealed the presence of oligomers with higher molecular weights compared to that of almonds. Copper ions were most effectively chelated by the constituents of the tannin fractions of hazelnuts, walnuts and almonds. At a 0.2 mg/assay addition level, the walnut tannins complexed almost 100% Cu(II). The Fe(II) complexation capacities of the tannin fractions of walnuts and hazelnuts were weaker in comparison to that of the almond tannin fraction, which at a 2.5 mg/assay addition level, bound Fe(II) by ~90%. The capacity to chelate Zn(II) was quite varied for the different nut tannin fractions: almond tannins bound as much as 84% Zn(II), whereas the value for walnut tannins was only 8.7%; and for hazelnut tannins, no Zn(II) chelation took place at the levels tested. PMID:20054482

Zn(II)-Coordinated Quantum Dot-FRET Nanosensors for the Detection of Protein Kinase Activity.

PubMed

Lim, Butaek; Park, Ji-In; Lee, Kyung Jin; Lee, Jin-Won; Kim, Tae-Wuk; Kim, Young-Pil

2015-07-23

We report a simple detection of protein kinase activity using Zn(II)-mediated fluorescent resonance energy transfer (FRET) between quantum dots (QDs) and dye-tethered peptides. With neither complex chemical ligands nor surface modification of QDs, Zn(II) was the only metal ion that enabled the phosphorylated peptides to be strongly attached on the carboxyl groups of the QD surface via metal coordination, thus leading to a significant FRET efficiency. As a result, protein kinase activity in intermixed solution was efficiently detected by QD-FRET via Zn(II) coordination, especially when the peptide substrate was combined with affinity-based purification. We also found that mono- and di-phosphorylation in the peptide substrate could be discriminated by the Zn(II)-mediated QD-FRET. Our approach is expected to find applications for studying physiological function and signal transduction with respect to protein kinase activity.

CdS/TiO2-fluorescein isothiocyanate nanoparticles as fluorescence resonance energy transfer probe for the determination of trace alkaline phosphatase based on affinity adsorption assay.

PubMed

Liu, Jia-Ming; Lin, Li-ping; Jiao, Li; Cui, Ma-Lin; Wang, Xin-Xing; Zhang, Li-Hong; Zheng, Zhi-Yong

2012-08-30

The CdS/TiO(2)-fluorescein isothiocyanate (FITC) luminescent nanoparticles (CdS/TiO(2)-FITC) with the particle size of 20 nm have been synthesized by sol-gel method. CdS/TiO(2)-FITC could emit the fluorescence of both FITC and CdS/TiO(2). The fluorescence resonance energy transfer (FRET) occurred between the donor CdS/TiO(2) and the acceptor FITC in the CdS/TiO(2)-FITC. Taking advantages of the excellent characteristics of FRET, a new CdS/TiO(2)-FITC FRET labeling reagent and a CdS/TiO(2)-FITC-wheat germ agglutinin (CdS/TiO(2)-FITC-WGA) fluorescent probe have been developed. The FRET occurring between the donor CdS/TiO(2) and the acceptor FITC in the labelled product CdS/TiO(2)-FITC-WGA-AP, formed in the affinity adsorption reaction between the WGA in this CdS/TiO(2)-FITC-WGA fluorescent probe and alkaline phosphatase (AP), sharply enhanced the fluorescence signal of FITC and quench the fluorescence signal of CdS/TiO(2). Moreover, the ΔF (the change of the fluorescence signal) of FITC and CdS/TiO(2) were proportional to the content of AP, respectively. Thus, a new method that CdS/TiO(2)-fluorescein isothiocyanate nanoparticles for the determination of trace AP based on FRET-affinity adsorption assay has been established. The limit of quantification (LOQ) of the method was 1.3×10(-17) g AP mL(-1) for CdS/TiO(2) and 1.1×10(-17) g AP mL(-1) for FITC, respectively. This sensitive, rapid, high selective and precise method has been applied to the determination of AP in human serum and the prediction of human disease with the results agreed well with enzyme-linked immunosorbent assay (ELISA) in Zhangzhou Municipal Hospital of Fujian Province. Simultaneously, the reaction mechanism for the determination of AP was also discussed. Copyright © 2012 Elsevier B.V. All rights reserved.

Synthesis, characterization, photoluminescence, and electrochemical studies of novel mononuclear Cu(II) and Zn(II) complexes with the 1-benzylimidazolium ligand

NASA Astrophysics Data System (ADS)

Bibi, Sherino; Mohammad, Sharifah; Manan, Ninie Suhana Abdul; Ahmad, Jimmy; Kamboh, Muhammad Afzal; Khor, Sook Mei; Yamin, Bohari M.; Abdul Halim, Siti Nadiah

2017-08-01

Two new mononuclear coordination complexes [Cu(bim)4Cl2]ṡ2H2O (1) and [Zn(bim)2Cl2] (2) containing the 1-benzylimidazole (bim) ligand were successfully synthesized. Both complexes were characterized by IR, UV-vis, and fluorescence spectroscopies, single crystal and powder X-ray diffraction measurements, and thermogravimetric analysis. Self-assembly during the recrystallization process resulted in the formation of octahedral and tetrahedral Cu(II) and Zn(II) complexes, respectively. The single crystals obtained are representative of the bulk material, as shown by the powder X-ray diffraction patterns. Cyclic voltammetry measurements showed that complex 1 undergoes a quasi-reversible redox reaction, while complex 2 undergoes reduction alone, and no oxidation peak was observed; this is due to the stability of the reduced form of complex 2.

Zn(II)-Coordinated Quantum Dot-FRET Nanosensors for the Detection of Protein Kinase Activity

PubMed Central

Lim, Butaek; Park, Ji-In; Lee, Kyung Jin; Lee, Jin-Won; Kim, Tae-Wuk; Kim, Young-Pil

2015-01-01

We report a simple detection of protein kinase activity using Zn(II)-mediated fluorescent resonance energy transfer (FRET) between quantum dots (QDs) and dye-tethered peptides. With neither complex chemical ligands nor surface modification of QDs, Zn(II) was the only metal ion that enabled the phosphorylated peptides to be strongly attached on the carboxyl groups of the QD surface via metal coordination, thus leading to a significant FRET efficiency. As a result, protein kinase activity in intermixed solution was efficiently detected by QD-FRET via Zn(II) coordination, especially when the peptide substrate was combined with affinity-based purification. We also found that mono- and di-phosphorylation in the peptide substrate could be discriminated by the Zn(II)-mediated QD-FRET. Our approach is expected to find applications for studying physiological function and signal transduction with respect to protein kinase activity. PMID:26213934

Structural basis of Zn(II) induced metal detoxification and antibiotic resistance by histidine kinase CzcS in Pseudomonas aeruginosa

PubMed Central

Wang, Dan; Chen, Weizhong; Huang, Shanqing; He, Yafeng; Liu, Xichun; Hu, Qingyuan; Wei, Tianbiao; Sang, Hong; Gan, Jianhua

2017-01-01

Pseudomonas aeruginosa (P. aeruginosa) is a major opportunistic human pathogen, causing serious nosocomial infections among immunocompromised patients by multi-determinant virulence and high antibiotic resistance. The CzcR-CzcS signal transduction system in P. aeruginosa is primarily involved in metal detoxification and antibiotic resistance through co-regulating cross-resistance between Zn(II) and carbapenem antibiotics. Although the intracellular regulatory pathway is well-established, the mechanism by which extracellular sensor domain of histidine kinase (HK) CzcS responds to Zn(II) stimulus to trigger downstream signal transduction remains unclear. Here we determined the crystal structure of the CzcS sensor domain (CzcS SD) in complex with Zn(II) at 1.7 Å resolution. This is the first three-dimensional structural view of Zn(II)-sensor domain of the two-component system (TCS). The CzcS SD is of α/β-fold in nature, and it senses the Zn(II) stimulus at micromole level in a tetrahedral geometry through its symmetry-related residues (His55 and Asp60) on the dimer interface. Though the CzcS SD resembles the PhoQ-DcuS-CitA (PDC) superfamily member, it interacts with the effector in a novel domain with the N-terminal α-helices rather than the conserved β-sheets pocket. The dimerization of the N-terminal H1 and H1’ α-helices is of primary importance for the activity of HK CzcS. This study provides preliminary insight into the molecular mechanism of Zn(II) sensing and signaling transduction by the HK CzcS, which will be beneficial to understand how the pathogen P. aeruginosa resists to high levels of heavy metals and antimicrobial agents. PMID:28732057

Structural basis of Zn(II) induced metal detoxification and antibiotic resistance by histidine kinase CzcS in Pseudomonas aeruginosa.

PubMed

Wang, Dan; Chen, Weizhong; Huang, Shanqing; He, Yafeng; Liu, Xichun; Hu, Qingyuan; Wei, Tianbiao; Sang, Hong; Gan, Jianhua; Chen, Hao

2017-07-01

Pseudomonas aeruginosa (P. aeruginosa) is a major opportunistic human pathogen, causing serious nosocomial infections among immunocompromised patients by multi-determinant virulence and high antibiotic resistance. The CzcR-CzcS signal transduction system in P. aeruginosa is primarily involved in metal detoxification and antibiotic resistance through co-regulating cross-resistance between Zn(II) and carbapenem antibiotics. Although the intracellular regulatory pathway is well-established, the mechanism by which extracellular sensor domain of histidine kinase (HK) CzcS responds to Zn(II) stimulus to trigger downstream signal transduction remains unclear. Here we determined the crystal structure of the CzcS sensor domain (CzcS SD) in complex with Zn(II) at 1.7 Å resolution. This is the first three-dimensional structural view of Zn(II)-sensor domain of the two-component system (TCS). The CzcS SD is of α/β-fold in nature, and it senses the Zn(II) stimulus at micromole level in a tetrahedral geometry through its symmetry-related residues (His55 and Asp60) on the dimer interface. Though the CzcS SD resembles the PhoQ-DcuS-CitA (PDC) superfamily member, it interacts with the effector in a novel domain with the N-terminal α-helices rather than the conserved β-sheets pocket. The dimerization of the N-terminal H1 and H1' α-helices is of primary importance for the activity of HK CzcS. This study provides preliminary insight into the molecular mechanism of Zn(II) sensing and signaling transduction by the HK CzcS, which will be beneficial to understand how the pathogen P. aeruginosa resists to high levels of heavy metals and antimicrobial agents.

The Effect of Hydrogen Bonding in Enhancing the Ionic Affinities of Immobilized Monoprotic Phosphate Ligands

DOE PAGES

Alexandratos, Spiro D.; Zhu, Xiaoping

2017-08-18

Environmental remediation requires ion-selective polymers that operate under a wide range of solution conditions. In one example, removal of trivalent and divalent metal ions from waste streams resulting from mining operations before they enter the environment requires treatment at acidic pH. The monoethyl ester phosphate ligands developed in this report operate from acidic solutions. They have been prepared on polystyrene-bound ethylene glycol, glycerol, and pentaerythritol, and it is found that intra-ligand hydrogen bonding affects their metal ion affinities. The affinity for a set of trivalent (Fe(III), Al(III), La(III), and Lu(III)) and divalent (Pb(II), Cd(II), Cu(II), and Zn(II)) ions is greatermore » than that of corresponding neutral diethyl esters and phosphonic acid. In an earlier study, hydrogen bonding was found important in determining the metal ion affinities of immobilized phosphorylated polyol diethyl ester coordinating ligands; their Fourier transform infrared (FTIR) band shifts indicated that the basicity of the phosphoryl oxygen increased by hydrogen bonding to auxiliary –OH groups on the neighboring polyol. The same mechanism is operative with the monoprotic resins along with hydrogen bonding to the P–OH acid site. This is reflected in the FTIR spectra: the neutral phosphate diethyl ester resins have the P=O band at 1265 cm -1 while the monoethyl ester resins have the band shifted to 1230 cm -1; hydrogen bonding is further indicated by the broadness of this region down to 900 cm -1. Of the polymers studied, monoprotic pentaerythritol has the highest metal ion affinities.« less

The Effect of Hydrogen Bonding in Enhancing the Ionic Affinities of Immobilized Monoprotic Phosphate Ligands

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alexandratos, Spiro D.; Zhu, Xiaoping

Environmental remediation requires ion-selective polymers that operate under a wide range of solution conditions. In one example, removal of trivalent and divalent metal ions from waste streams resulting from mining operations before they enter the environment requires treatment at acidic pH. The monoethyl ester phosphate ligands developed in this report operate from acidic solutions. They have been prepared on polystyrene-bound ethylene glycol, glycerol, and pentaerythritol, and it is found that intra-ligand hydrogen bonding affects their metal ion affinities. The affinity for a set of trivalent (Fe(III), Al(III), La(III), and Lu(III)) and divalent (Pb(II), Cd(II), Cu(II), and Zn(II)) ions is greatermore » than that of corresponding neutral diethyl esters and phosphonic acid. In an earlier study, hydrogen bonding was found important in determining the metal ion affinities of immobilized phosphorylated polyol diethyl ester coordinating ligands; their Fourier transform infrared (FTIR) band shifts indicated that the basicity of the phosphoryl oxygen increased by hydrogen bonding to auxiliary –OH groups on the neighboring polyol. The same mechanism is operative with the monoprotic resins along with hydrogen bonding to the P–OH acid site. This is reflected in the FTIR spectra: the neutral phosphate diethyl ester resins have the P=O band at 1265 cm -1 while the monoethyl ester resins have the band shifted to 1230 cm -1; hydrogen bonding is further indicated by the broadness of this region down to 900 cm -1. Of the polymers studied, monoprotic pentaerythritol has the highest metal ion affinities.« less

Synthesis, structure, and characterization of two Zn(II) complex containing two-dimensional bilayer structure

NASA Astrophysics Data System (ADS)

Zhang, Meili; Ren, Yixia; Chen, Xiaoli

2014-10-01

Two new Zn(II) complexes, [Zn2(L)(H2O)3]ṡH2O (1) and [Zn3(HL)2(bpp)2(Hbpp)2]ṡ10H2Oṡ2ClO4 (2) (H4L = cis,cis,cis,cis-1,2,3,4-cyclopentanetracarboxylic acid, bpp = 1,3-bis(4-pyridyl)propane), have been synthesized and characterized by elemental analysis, IR spectroscopy and single-crystal X-ray diffraction techniques. The structure indicates that the complex 1 crystallizes in triclinic, space group Pī, in which, the four carboxylate groups of L ligand adopt μ2-η1:η0, μ2-η1:η1, μ1-η1:η1 coordination modes, respectively, bridging Zn(II) atoms to generate a (4,6)-connected 2D bilayer network. The structure indicates that the complex 2 crystallizes in monoclinic, space group C2/c, in which, three deprotonated carboxylate groups of L ligand adopt uniform μ1-η1:η0 coordination mode linking Zn(II) atoms to form a 1D polymeric ribbon, the bpp ligands further extend such ribbon giving rised to a (3,4)-connected 2D bilayer network. The most striking feature of 1 and 2 is that both of bilayer networks contain 1D solvent channel, where water molecules are located. In additional, luminescent properties of two complexes have also been studied.

Determination of aflatoxins and ochratoxin A in high-sugar-content traditional Turkish foods by affinity column cleanup and LC fluorescence detection.

PubMed

Senyuva, Hamide Z; Cimen, Dilek; Gilbert, John

2009-01-01

The effectiveness of an affinity column cleanup procedure followed by LC with fluorescence detection was established for the determination of aflatoxins and ochratoxin A in high-sugar-content traditional Turkish foods. Traditional foods, such as baklava (finely layered pastry filled with nuts and steeped in syrup), halvah (containing sesame paste and pistachios), cevizli sucuk (a confection made of grape juice boiled and dried on strings of nuts), Turkish delight (containing hazelnuts, pistachios, or walnuts), and pişmaniye (candy made of sugar, butter, and flour), were tested, and the performance of the method was established with spiked samples. To examine the robustness of the methodology, baklava was prepared from raw materials and spiked at the initial stage of dry ingredients and through subsequent stages of preparation of dough, after cooking, and after addition of syrup and nuts. For all products, the analytical method required grinding the composite foodstuff under liquid nitrogen to form a fine powder, which was then thoroughly mixed before subsampling. After vortex extraction into methanol-water (aflatoxins) and aqueous sodium bicarbonate (ochratoxin A), the sample was filtered, diluted with phosphate-buffered saline, and then passed through either an aflatoxin or ochratoxin A affinity column before HPLC analysis with fluorescence detection (using post-column bromination for the aflatoxins). In all the traditional Turkish products, the recovery of aflatoxin B1 ranged from 77 to 98%, and LODs were <0.1 microg/kg. For ochratoxin A, the recoveries were from 88 to 93% and LODs were similarly <0.1 microLg/kg. Despite the complex nature of these traditional Turkish foods, which frequently contain products from sugar caramelization, there was no evidence of any interfering co-extractives, and the method has proved to be robust enough to be used for food control purposes.

Effect of biofilm coatings at metal-oxide/water interfaces II: Competitive sorption between Pb(II) and Zn(II) at Shewanella oneidensis/metal-oxide/water interfaces

DOE PAGES

Wang, Yingge; Gelabert, Alexandre; Michel, F. Marc; ...

2016-05-07

Competitive sorption of Pb(II) and Zn(II) on Shewanella oneidensis MR-1 biofilm-coated single-crystal α-Al 2O 3 (1 –1 0 2) and α-Fe 2O 3 (0 0 0 1) surfaces was investigated using long-period X-ray standing wave-florescence yield (LP-XSW-FY) spectroscopy. In situ partitioning of aqueous Pb(II) and Zn(II) between the biofilms and underlying metal-oxide substrates was probed following exposure of these complex interfaces to equi-molar Pb and Zn solutions (0.01 M NaNO 3 as background electrolyte, pH = 6.0, and 3-h equilibration time). At higher Pb and Zn concentrations (≥10 –5 M), more than 99% of these ions partitioned into the biofilmsmore » at S. oneidensis/α-Al 2O 3 (1 –1 0 2)/water interfaces, which is consistent with the partitioning behavior of both Pb(II) or Zn(II) in single-metal-ion experiments. Furthermore, no apparent competitive effects were found in this system at these relatively high metal-ion concentrations. However, at lower equi-molar concentrations (≤10 –6 M), Pb(II) and Zn(II) partitioning in the same system changed significantly compared to the single-metal-ion systems. The presence of Zn(II) decreased Pb(II) partitioning onto α-Al 2O 3 (1 –1 0 2) substantially (~52% to ~13% at 10 –7 M, and ~23% to ~5% at 10–6 M), whereas the presence of Pb(II) caused more Zn(II) to partition onto α-Al 2O 3 (1 –1 0 2) surfaces (~15% to ~28% at 10 –7 M, and ~1% to ~7% at 10 –6 M) .The higher observed partitioning of Zn(II) (~28%) at the α-Al 2O 3 (1 –1 0 2) surfaces compared to Pb(II) (~13%) in the mixed-metal-ion systems at the lowest concentration (10 –7 M) suggests that Zn(II) is slightly favored over Pb(II) for sorption sites on α-Al 2O 3 (1 –1 0 2) surfaces under our experimental conditions.« less

Simultaneous Stripping Detection of Pb(II), Cd(II) and Zn(II) Using a Bimetallic Hg-Bi/Single-Walled Carbon Nanotubes Composite Electrode

PubMed Central

Ouyang, Ruizhuo; Zhu, Zhenqian; Tatum, Clarissa E.; Chambers, James Q.; Xue, Zi-Ling

2011-01-01

A new, sensitive platform for the simultaneous electrochemical assay of Zn(II), Cd(II) and Pb(II) in aqueous solution has been developed. The platform is based on a new bimetallic Hg-Bi/single-walled carbon nanotubes (SWNTs) composite modified glassy carbon electrode (GCE), demonstrating remarkably improved performance for the anodic stripping assay of Zn(II), Cd(II) and Pb(II). The synergistic effect of Hg and Bi as well as the enlarged, activated surface and good electrical conductivity of SWNTs on GCE contribute to the enhanced activity of the proposed electrode. The analytical curves for Zn(II), Cd(II) an Pb(II) cover two linear ranges varying from 0.5 to 11 μg L-1 and 10 to 130 μg L-1 with correlation coefficients higher than 0.992. The limits of detection for Zn(II), Cd(II) are lower than 2 μg L-1 (S/N = 3). For Pb(II), moreover, there is another lower, linear range from 5 to 1100 ng L-1 with a coefficient of 0.987 and a detection limit of 0.12 ng L-1. By using the standard addition method, Zn(II), Cd(II) and Pb(II) ions in river samples were successfully determined. These results suggest that the proposed method can be applied as a simple, efficient alternative for the simultaneous monitoring of heavy metals in water samples. In addition, this method demonstrates the powerful application of carbon nanotubes in electrochemical analysis of heavy metals. PMID:21660117

Crystal structures and thermodynamics/kinetics of Zn(II) coordination polymers with helical chains

DOE Office of Scientific and Technical Information (OSTI.GOV)

He, Tian; Yue, Ke-Fen, E-mail: ykflyy@nwu.edu.cn; Zhao, Yi-xing

Solvothermal reactions of Zn(II) acetates and four V-shaped carboxylates ligands in the presence of 1,4-Bis(2-methyl-imidazol-1-yl)butane afforded four interesting Zn(II) coordination polymers with helical chains, namely, {[Zn(bib)(atibdc)]·2H_2O}{sub n} (1), {[Zn(bib)(atbip)]·H_2O}{sub n} (2), {[Zn(bib)(2,2′-tda)]}{sub n} (3) and {[Zn(bib)(5-tbipa)]·EtOH}{sub n} (4), (H{sub 2}atibdc=5-amino-2,4,6-triiodoisophthalic acid, H{sub 2}atbip=5-amino-2,4,6-tribromoisophthalic acid, 2,2′-H{sub 2}tad=2,2′-thiodiacetic acid, 5-H{sub 2}tbipa=5-tert-butyl-isophthalic acid). 1 reveals a 3D chiral framework with three kinds of helical chains along a, b and c axis. 2 shows a 2D step-type chiral framework with right-handed helical chains. 3 displays a wavelike 2D layer network possessing alternate left- and right-handed helical chains. 4 presents a four-connected 3D framework withmore » zigzag and meso-helical chains. The different spacers and substituent group of carboxylic acid ligands may lead to the diverse network structures of 1–4. The fluorescent properties of complexes 1−4 were studied. In addition, the thermal decompositions properties of 1–4 were investigated by simultaneous TG/DTG–DSC technique. The apparent activation energy E and the pre-exponential factor (A) of skeleton collapse for the complexes 1–4 are calculated by the integral Kissinger's method and Ozawa–Doyle's method. The activation energy E (E{sub 1}=209.658 kJ·mol{sup −1}, E{sub 2}=250.037 kJ mol{sup −1}, E{sub 3}=225.300 kJ mol{sup −1}, E{sub 4}=186.529 kJ·mol{sup −1}) demonstrates that the reaction rate of the melting decomposition is slow. The thermodynamic parameters (ΔH{sup ‡}, ΔG{sup ‡} and ΔS{sup ‡}) at the peak temperatures of the DTG curves were also calculated. ΔG{sup ‡}>0 indicates that the skeleton collapse is not spontaneous. ΔH{sub d}>0 suggests that the skeleton collapse is endothermic, corresponding to the intense endothermic peak of the DSC

The ligand effect on the hydrolytic reactivity of Zn(II) complexes toward phosphate diesters.

PubMed

Bonfá, Lodovico; Gatos, Maddalena; Mancin, Fabrizio; Tecilla, Paolo; Tonellato, Umberto

2003-06-16

The catalytic effects of the Zn(II) complexes of a series of poliaminic ligands in the hydrolysis of the activated phosphodiesters bis-p-nitrophenyl phosphate (BNP) and 2-hydroxypropyl-p-nitrophenyl phosphate (HPNP) have been investigated. The reactions show first-order rate dependency on both substrate and metal ion complex and a pH dependence which is diagnostic of the acid dissociation of the reactive species. The mechanism of the metal catalyzed transesterification of HPNP has been assessed by solvent isotopic kinetic effect studies and involves the intramolecular nucleophilic attack of the substrate alcoholic group, activated by metal ion coordination. The intrinsic reactivity of the different complexes is controlled by the nature and structure of the ligand: complexes of tridentate ligands, particularly if characterized by a facial coordination mode, are more reactive than those of tetradentate ligands which can hardly allow binding sites for the substrate. In the case of tridentate ligands that form complexes with a facial coordination mode, a linear Brønsted correlation between the reaction rate (log k) and the pK(a) of the active nucleophile is obtained. The beta(nuc) values are 0.75 for the HPNP transesterification and 0.20 for the BNP hydrolysis. These values are indicated as the result of the combination of two opposite Lewis acid effects of the Zn(II) ion: the activation of the substrate and the efficiency of the metal coordinated nucleophile. The latter factor apparently prevails in determining the intrinsic reactivity of the Zn(II) complexes.

Neuroprotective Effects and Mechanisms of Curcumin-Cu(II) and -Zn(II) Complexes Systems and Their Pharmacological Implications.

PubMed

Yan, Fa-Shun; Sun, Jian-Long; Xie, Wen-Hai; Shen, Liang; Ji, Hong-Fang

2017-12-28

Alzheimer's disease (AD) is the main form of dementia and has a steadily increasing prevalence. As both oxidative stress and metal homeostasis are involved in the pathogenesis of AD, it would be interesting to develop a dual function agent, targeting the two factors. Curcumin, a natural compound isolated from the rhizome of Curcuma longa , is an antioxidant and can also chelate metal ions. Whether the complexes of curcumin with metal ions possess neuroprotective effects has not been evaluated. Therefore, the present study was designed to investigate the protective effects of the complexes of curcumin with Cu(II) or Zn(II) on hydrogen peroxide (H₂O₂)-induced injury and the underlying molecular mechanisms. The use of rat pheochromocytoma (PC12) cells, a widely used neuronal cell model system, was adopted. It was revealed that curcumin-Cu(II) complexes systems possessed enhanced O₂ ·- -scavenging activities compared to unchelated curcumin. In comparison with unchelated curcumin, the protective effects of curcumin-Cu(II) complexes systems were stronger than curcumin-Zn(II) system. Curcumin-Cu(II) or -Zn(II) complexes systems significantly enhanced the superoxide dismutase, catalase, and glutathione peroxidase activities and attenuated the increase of malondialdehyde levels and caspase-3 and caspase-9 activities, in a dose-dependent manner. The curcumin-Cu(II) complex system with a 2:1 ratio exhibited the most significant effect. Further mechanistic study demonstrated that curcumin-Cu(II) or -Zn(II) complexes systems inhibited cell apoptosis via downregulating the nuclear factor κB (NF-κB) pathway and upregulating Bcl-2/Bax pathway. In summary, the present study found that curcumin-Cu(II) or -Zn(II) complexes systems, especially the former, possess significant neuroprotective effects, which indicates the potential advantage of curcumin as a promising agent against AD and deserves further study.

Discovery of potent cytotoxic ortho-aryl chalcones as new scaffold targeting tubulin and mitosis with affinity-based fluorescence.

PubMed

Zhu, Cuige; Zuo, Yinglin; Wang, Ruimin; Liang, Baoxia; Yue, Xin; Wen, Gesi; Shang, Nana; Huang, Lei; Chen, Yu; Du, Jun; Bu, Xianzhang

2014-08-14

A series of new ortho-aryl chalcones have been designed and synthesized. Many of these compounds were found to exhibit significant antiproliferation activity toward a panel of cancer cell lines. Selected compounds show potent cytotoxicity against several drug resistant cell lines including paclitaxel (Taxol) resistant human ovarian carcinoma cells, vincristine resistant human ileocecum carcinoma cells, and doxorubicin resistant human breast carcinoma cells. Further investigation revealed that active analogues could inhibit the microtubule polymerization by binding to colchicine site and thus induce multipolar mitosis, G2/M phase arrest, and apoptosis of cancer cells. Furthermore, affinity-based fluorescence enhancement was observed during the binding of active compounds with tubulin, which greatly facilitated the determination of tubulin binding site of the compounds. Finally, selected compound 26 was found to exhibit obvious in vivo antitumor activity in A549 tumor xenografts model. Our systematic studies implied a new scaffold targeting tubulin and mitosis for novel antitumor drug discovery.

Fluorescence Analysis of Sulfonamide Binding to Carbonic Anhydrase

ERIC Educational Resources Information Center

Wang, Sheila C.; Zamble, Deborah B.

2006-01-01

A practical laboratory experiment is described that illustrates the application of fluorescence resonance energy transfer to the study of protein-ligand binding. The affinities of wild-type and mutant human carbonic anhydrase II for dansylamide were determined by monitoring the increase in ligand fluorescence that occurs due to energy transfer…

A saponification-triggered gelation of ester-based Zn(II) complex through conformational transformations.

PubMed

Kumar, Ashish; Dubey, Mrigendra; Kumar, Amit; Pandey, Daya Shankar

2014-09-11

Novel saponification-triggered gelation in an ester-based bis-salen Zn(II) complex (1) is described. Strategic structural modifications induced by NaOH in 1 tune the dipolar-/π-interactions leading to J-aggregation and the creation of an inorganic gel material (IGM), which has been established by photophysical, DFT and rheological studies.

Potential toxicity and affinity of triphenylmethane dye malachite green to lysozyme.

PubMed

Ding, Fei; Li, Xiu-Nan; Diao, Jian-Xiong; Sun, Ye; Zhang, Li; Ma, Lin; Yang, Xin-Ling; Zhang, Li; Sun, Ying

2012-04-01

Malachite green is a triphenylmethane dye that is used extensively in many industrial and aquacultural processes, generating environmental concerns and health problems to human being. In this contribution, the complexation between lysozyme and malachite green was verified by means of computer-aided molecular modeling, steady state and time-resolved fluorescence, and circular dichroism (CD) approaches. The precise binding patch of malachite green in lysozyme has been identified from molecular modeling and ANS displacement, Trp-62, Trp-63, and Trp-108 residues of lysozyme were earmarked to possess high-affinity for this dye, the principal forces in the lysozyme-malachite green adduct are hydrophobic and π-π interactions. Steady state fluorescence proclaimed the complex of malachite green with lysozyme yields quenching through static type, which substantiates time-resolved fluorescence measurements that lysozyme-malachite green conjugation formation has an affinity of 10(3)M(-1). Moreover, via molecular modeling and also CD data, we can safely arrive at a conclusion that the polypeptide chain of lysozyme partially destabilized upon complexation with malachite green. The data emerged here will help to further understand the toxicological action of malachite green in human body. Copyright © 2012 Elsevier Inc. All rights reserved.

Methods for quantifying T cell receptor binding affinities and thermodynamics

PubMed Central

Piepenbrink, Kurt H.; Gloor, Brian E.; Armstrong, Kathryn M.; Baker, Brian M.

2013-01-01

αβ T cell receptors (TCRs) recognize peptide antigens bound and presented by class I or class II major histocompatibility complex (MHC) proteins. Recognition of a peptide/MHC complex is required for initiation and propagation of a cellular immune response, as well as the development and maintenance of the T cell repertoire. Here we discuss methods to quantify the affinities and thermodynamics of interactions between soluble ectodomains of TCRs and their peptide/MHC ligands, focusing on titration calorimetry, surface plasmon resonance, and fluorescence anisotropy. As TCRs typically bind ligand with weak-to-moderate affinities, we focus the discussion on means to enhance the accuracy and precision of low affinity measurements. In addition to further elucidating the biology of the T cell mediated immune response, more reliable low affinity measurements will aid with more probing studies with mutants or altered peptides that can help illuminate the physical underpinnings of how TCRs achieve their remarkable recognition properties. PMID:21609868

Zn(II) coordination polymers with flexible V-shaped dicarboxylate ligand: Syntheses, helical structures and properties

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Lin; Liu, Chong-Bo, E-mail: cbliu@nchu.edu.cn; Yang, Gao-Shan

2015-11-15

Hydrothermal reactions of 2,2′-[hexafluoroisopropylidenebis(p-phenyleneoxy)]diacetic acid (H{sub 2}L) and zinc ions in the presence of N-donor ancillary ligands afford four novel coordination polymers, namely, [Zn{sub 2}(μ{sub 2}-OH)(μ{sub 4}-O){sub 0.5}(L)]·0.5H{sub 2}O (1), [Zn(L)(2,2′-bipy)(H{sub 2}O)] (2), [Zn{sub 3}(L){sub 3}(phen){sub 2}]·H{sub 2}O (3) and [Zn{sub 2}(L){sub 2}(4,4′-bipy)] (4) (2,2′-bipy=2,2′-bipyridine; 4,4′-bipy=4,4′-bipyridine; phen=1,10-phenanthroline). Their structures have been determined by single-crystal X-ray diffraction analyses, elemental analyses, IR spectra, powder X-ray diffraction (PXRD), and thermogravimetric (TG) analyses. Complex 1 shows a 3-D clover framework consisting of [Zn{sub 4}(µ{sub 4}-O)(µ{sub 2}-OH){sub 2}]{sup 4+} clusters, and exhibits a novel (3,8)-connected topological net with the Schläfli symbol of {3·4·5}{sub 2}{3"4·4"4·5"2·6"6·7"1"0·8"2}, andmore » contains double-stranded and two kinds of meso-helices. 2 displays a helical chain structure, which is further extended via hydrogen bonds into a 3-D supramolecular structure with meso-helix chains. 3 displays a 2-D {4"4·6"2} parallelogram structure, which is further extended via hydrogen bonds into a 3-D supramolecular structure with single-stranded helical chains. 4 shows a 2-D {4"4·6"2} square structure with left- and right-handed helical chains. Moreover, the luminescent properties of 1–4 have been investigated. - Graphical abstract: Four new Zn(II) coordination polymers with helical structures based on flexible V-shaped dicarboxylate ligand have been synthesized and structurally characterized. Photoluminescent properties have been investigated. - Highlights: • Four novel Zn(II) coordination polymers with V-shaped ligand were characterized. • Complexes 1–4 show diverse intriguing helical characters. • Fluorescence properties of complexes 1–4 were investigated.« less

Possible steric control of the relative strength of chelation enhanced fluorescence for zinc(II) compared to cadmium(II): metal ion complexing properties of tris(2-quinolylmethyl)amine, a crystallographic, UV-visible, and fluorometric study.

PubMed

Williams, Neil J; Gan, Wei; Reibenspies, Joseph H; Hancock, Robert D

2009-02-16

The idea is examined that steric crowding in ligands can lead to diminution of the chelation enhanced fluorescence (CHEF) effect in complexes of the small Zn(II) ion as compared to the larger Cd(II) ion. Steric crowding is less severe for the larger ion and for the smaller Zn(II) ion leads to Zn-N bond length distortion, which allows some quenching of fluorescence by the photoinduced electron transfer (PET) mechanism. Some metal ion complexing properties of the ligand tris(2-quinolylmethyl)amine (TQA) are presented in support of the idea that more sterically efficient ligands, which lead to less M-N bond length distortion with the small Zn(II) ion, will lead to a greater CHEF effect with Zn(II) than Cd(II). The structures of [Zn(TQA)H(2)O](ClO(4))(2).1.5 H(2)O (1), ([Pb(TQA)(NO(3))(2)].C(2)H(5)OH) (2), ([Ag(TQA)(ClO(4))]) (3), and (TQA).C(2)H(5)OH (4) are reported. In 1, the Zn(II) is 5-coordinate, with four N-donors from the ligand and a water molecule making up the coordination sphere. The Zn-N bonds are all of normal length, showing that the level of steric crowding in 1 is not sufficient to cause significant Zn-N bond length distortion. This leads to the observation that, as expected, the CHEF effect in the Zn(II)/TQA complex is much stronger than that in the Cd(II)/TQA complex, in contrast to similar but more sterically crowded ligands, where the CHEF effect is stronger in the Cd(II) complex. The CHEF effect for TQA with the metal ions examined varies as Zn(II) > Cd(II) > Ni(II) > Pb(II) > Hg(II) > Cu(II). The structure of 2 shows an 8-coordinate Pb(II), with evidence of a stereochemically active lone pair, and normal Pb-N bond lengths. In 3, the Ag(I) is 5-coordinate, with four N-donors from the TQA and an oxygen from the perchlorate. The Ag(I) shows no distortion toward linear 2-coordinate geometry, and the Ag-N bonds fall slightly into the upper range for Ag-N bonds in 5-coordinate complexes. The structure of 4 shows the TQA ligand to be involved in pi

Synthesis, characterization, and application of cy-dye- and alexa-dye-labeled hongotoxin(1) analogues. The first high affinity fluorescence probes for voltage-gated K+ channels.

PubMed

Pragl, Bernt; Koschak, Alexandra; Trieb, Maria; Obermair, Gerald; Kaufmann, Walter A; Gerster, Uli; Blanc, Eric; Hahn, Christoph; Prinz, Heino; Schütz, Gerhard; Darbon, Herve; Gruber, Hermann J; Knaus, Hans-Günther

2002-01-01

Hongotoxin(1) (HgTX(1)), a 39-residue peptide recently isolated from the venom of Centruroides limbatus, blocks the voltage-gated K+ channels K(v)1.1, K(v)1.2, and K(v)1.3 at picomolar toxin concentrations (Koschak, A., Bugianesi, R. M., Mitterdorfer, J., Kaczorowski, G. J., Garcia, M. L., and Knaus, H. G. (1998) J. Biol. Chem. 273, 2639-2644). In this report, we determine the three-dimensional structure of HgTX(1) using NMR spectroscopy (PDB-code: 1HLY). HgTX(1) was found to possess a structure similar to previously characterized K+ channel toxins (e.g. margatoxin) consisting of a three-stranded antiparallel beta-sheet (residues 2-4, 26-30, and 33-37) and a helical conformation (part 3(10) helix and part alpha helix; residues 10-20). Due to the importance of residue Lys-28 for high-affinity interaction with the respective channels, lysine-reactive fluorescence dyes cannot be used to label wild-type HgTX(1). On the basis of previous studies (see above) and our NMR data, a HgTX(1) mutant (HgTX(1)-A19C) was engineered, expressed, and purified. HgTX(1)-A19C-SH was labeled using sulfhydryl-reactive Cy3-, Cy5-, and Alexa-dyes. Pharmacological characterization of fluorescently labeled HgTX(1)-A19C in radioligand binding studies indicated that these hongotoxin(1) analogues retain high-affinity for voltage-gated K+ channels and a respective pharmacological profile. Cy3- and Alexa-dye-labeled hongotoxin(1) analogues were used to investigate the localization of K+ channels in brain sections. The distribution of toxin binding closely follows the distribution of K(v)1.2 immunoreactivity with the highest expression levels in the cerebellar Purkinje cell layer. Taken together, these results demonstrate that fluorescently labeled HgTX(1) analogues comprise novel probes to characterize a subset of voltage-gated K+ channels.

RNA aptamers that functionally interact with green fluorescent protein and its derivatives

PubMed Central

Shui, Bo; Ozer, Abdullah; Zipfel, Warren; Sahu, Nevedita; Singh, Avtar; Lis, John T.; Shi, Hua; Kotlikoff, Michael I.

2012-01-01

Green Fluorescent Protein (GFP) and related fluorescent proteins (FPs) have been widely used to tag proteins, allowing their expression and subcellular localization to be examined in real time in living cells and animals. Similar fluorescent methods are highly desirable to detect and track RNA and other biological molecules in living cells. For this purpose, we have developed a group of RNA aptamers that bind GFP and related proteins, which we term Fluorescent Protein-Binding Aptamers (FPBA). These aptamers bind GFP, YFP and CFP with low nanomolar affinity and binding decreases GFP fluorescence, whereas slightly augmenting YFP and CFP brightness. Aptamer binding results in an increase in the pKa of EGFP, decreasing the 475 nm excited green fluorescence at a given pH. We report the secondary structure of FPBA and the ability to synthesize functional multivalent dendrimers. FPBA expressed in live cells decreased GFP fluorescence in a valency-dependent manner, indicating that the RNA aptamers function within cells. The development of aptamers that bind fluorescent proteins with high affinity and alter their function, markedly expands their use in the study of biological pathways. PMID:22189104

Affinity interactions between natural pigments and human whole saliva.

PubMed

Yao, Jiang-Wu; Lin, Feng; Tao, Tao; Lin, Chang-Jian

2011-03-01

The aim of the present study was to assess the null hypothesis that there are no differences of affinity between pigments and human whole saliva (WS), and the affinity is not influenced by the functional groups of pigments, temperatures, pH values, and salt concentrations. The affinity constants of interactions between WS and theaflavin (TF)/curcumin (Cur)/cyanidin (Cy) were determined by surface plasmon resonance (SPR) and fluorescence quenching. Mass-uptake at various temperatures, pH values, and salt concentrations was also carried out. The order of affinity of the pigments binding to WS is TF>Cur>Cy. A large number of complexes and precipitations of pigments/proteins were formed through a quick, strong, and almost irreversible binding process. The mass-uptake of pigments was affected not only by the functional groups, but also by molecular weight of pigments, temperatures, pH values, and salt concentrations. The complex of pigments may easily and rapidly deposit onto the WS film, and are difficult to remove from the WS surface. However, the complex of pigments can be reduced by properly regulating the physicochemical conditions, such as temperatures, pH values, and salt concentrations. Copyright © 2010 Elsevier Ltd. All rights reserved.

Earthworm Lumbricus rubellus MT-2: Metal Binding and Protein Folding of a True Cadmium-MT.

PubMed

Kowald, Gregory R; Stürzenbaum, Stephen R; Blindauer, Claudia A

2016-01-05

Earthworms express, as most animals, metallothioneins (MTs)-small, cysteine-rich proteins that bind d(10) metal ions (Zn(II), Cd(II), or Cu(I)) in clusters. Three MT homologues are known for Lumbricus rubellus, the common red earthworm, one of which, wMT-2, is strongly induced by exposure of worms to cadmium. This study concerns composition, metal binding affinity and metal-dependent protein folding of wMT-2 expressed recombinantly and purified in the presence of Cd(II) and Zn(II). Crucially, whilst a single Cd₇wMT-2 species was isolated from wMT-2-expressing E. coli cultures supplemented with Cd(II), expressions in the presence of Zn(II) yielded mixtures. The average affinities of wMT-2 determined for either Cd(II) or Zn(II) are both within normal ranges for MTs; hence, differential behaviour cannot be explained on the basis of overall affinity. Therefore, the protein folding properties of Cd- and Zn-wMT-2 were compared by ¹H NMR spectroscopy. This comparison revealed that the protein fold is better defined in the presence of cadmium than in the presence of zinc. These differences in folding and dynamics may be at the root of the differential behaviour of the cadmium- and zinc-bound protein in vitro, and may ultimately also help in distinguishing zinc and cadmium in the earthworm in vivo.

Two luminescent Zn(II) metal-organic frameworks for exceptionally selective detection of picric acid explosives.

PubMed

Shi, Zhi-Qiang; Guo, Zi-Jian; Zheng, He-Gen

2015-05-14

Two luminescent Zn(II) metal-organic frameworks were prepared from a π-conjugated thiophene-containing carboxylic acid ligand. These two MOFs show strong luminescene and their luminescence could be quenched by a series of nitroaromatic explosives. Importantly, they exhibit very highly sensitive and selective detection of picric acid compared to other nitroaromatic explosives.

Calcium binding to calmodulin mutants monitored by domain-specific intrinsic phenylalanine and tyrosine fluorescence.

PubMed Central

VanScyoc, Wendy S; Sorensen, Brenda R; Rusinova, Elena; Laws, William R; Ross, J B Alexander; Shea, Madeline A

2002-01-01

Cooperative calcium binding to the two homologous domains of calmodulin (CaM) induces conformational changes that regulate its association with and activation of numerous cellular target proteins. Calcium binding to the pair of high-affinity sites (III and IV in the C-domain) can be monitored by observing calcium-dependent changes in intrinsic tyrosine fluorescence intensity (lambda(ex)/lambda(em) of 277/320 nm). However, calcium binding to the low-affinity sites (I and II in the N-domain) is more difficult to measure with optical spectroscopy because that domain of CaM does not contain tryptophan or tyrosine. We recently demonstrated that calcium-dependent changes in intrinsic phenylalanine fluorescence (lambda(ex)/lambda(em) of 250/280 nm) of an N-domain fragment of CaM reflect occupancy of sites I and II (VanScyoc, W. S., and M. A. Shea, 2001, Protein Sci. 10:1758-1768). Using steady-state and time-resolved fluorescence methods, we now show that these excitation and emission wavelength pairs for phenylalanine and tyrosine fluorescence can be used to monitor equilibrium calcium titrations of the individual domains in full-length CaM. Calcium-dependent changes in phenylalanine fluorescence specifically indicate ion occupancy of sites I and II in the N-domain because phenylalanine residues in the C-domain are nonemissive. Tyrosine emission from the C-domain does not interfere with phenylalanine fluorescence signals from the N-domain. This is the first demonstration that intrinsic fluorescence may be used to monitor calcium binding to each domain of CaM. In this way, we also evaluated how mutations of two residues (Arg74 and Arg90) located between sites II and III can alter the calcium-binding properties of each of the domains. The mutation R74A caused an increase in the calcium affinity of sites I and II in the N-domain. The mutation R90A caused an increase in calcium affinity of sites III and IV in the C-domain whereas R90G caused an increase in calcium affinity

Batch affinity adsorption of His-tagged proteins with EDTA-based chitosan.

PubMed

Hua, Weiwei; Lou, Yimin; Xu, Weiyuan; Cheng, Zhixian; Gong, Xingwen; Huang, Jianying

2016-01-01

Affinity adsorption purification of hexahistidine-tagged (His-tagged) proteins using EDTA-chitosan-based adsorption was designed and carried out. Chitosan was elaborated with ethylenediaminetetraacetic acid (EDTA), and the resulting polymer was characterized by FTIR, TGA, and TEM. Different metals including Ni(2+), Cu(2+), and Zn(2+) were immobilized with EDTA-chitosan, and their capability to the specific adsorption of His-tagged proteins were then investigated. The results showed that Ni(2+)-EDTA-chitosan and Zn(2+)-EDTA-chitosan had high affinity toward the His-tagged proteins, thus isolating them from protein mixture. The target fluorescent-labeled hexahistidine protein remained its fluorescent characteristic throughout the purification procedure when Zn(2+)-EDTA-chitosan was used as a sorbent, wherein the real-time monitor was performed to examine the immigration of fluorescent-labeled His-tagged protein. Comparatively, Zn(2+)-EDTA-chitosan showed more specific binding ability for the target protein, but with less binding capacity. It was further proved that this purification system could be recovered and reused at least for 5 times and could run on large scales. The presented M(2+)-EDTA-chitosan system, with the capability to specifically bind His-tagged proteins, make the purification of His-tagged proteins easy to handle, leaving out fussy preliminary treatment, and with the possibility of continuous processing and a reduction in operational cost in relation to the costs of conventional processes.

Comprehensive analysis of RNA-protein interactions by high-throughput sequencing-RNA affinity profiling.

PubMed

Tome, Jacob M; Ozer, Abdullah; Pagano, John M; Gheba, Dan; Schroth, Gary P; Lis, John T

2014-06-01

RNA-protein interactions play critical roles in gene regulation, but methods to quantitatively analyze these interactions at a large scale are lacking. We have developed a high-throughput sequencing-RNA affinity profiling (HiTS-RAP) assay by adapting a high-throughput DNA sequencer to quantify the binding of fluorescently labeled protein to millions of RNAs anchored to sequenced cDNA templates. Using HiTS-RAP, we measured the affinity of mutagenized libraries of GFP-binding and NELF-E-binding aptamers to their respective targets and identified critical regions of interaction. Mutations additively affected the affinity of the NELF-E-binding aptamer, whose interaction depended mainly on a single-stranded RNA motif, but not that of the GFP aptamer, whose interaction depended primarily on secondary structure.

PDZ affinity chromatography: a general method for affinity purification of proteins based on PDZ domains and their ligands.

PubMed

Walkup, Ward G; Kennedy, Mary B

2014-06-01

PDZ (PSD-95, DiscsLarge, ZO1) domains function in nature as protein binding domains within scaffold and membrane-associated proteins. They comprise ∼90 residues and make specific, high affinity interactions with complementary C-terminal peptide sequences, with other PDZ domains, and with phospholipids. We hypothesized that the specific, strong interactions of PDZ domains with their ligands would make them well suited for use in affinity chromatography. Here we describe a novel affinity chromatography method applicable for the purification of proteins that contain PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We created a series of affinity resins comprised of PDZ domains from the scaffold protein PSD-95, or from neuronal nitric oxide synthase (nNOS), coupled to solid supports. We used them to purify heterologously expressed neuronal proteins or protein domains containing endogenous PDZ domain ligands, eluting the proteins with free PDZ domain peptide ligands. We show that Proteins of Interest (POIs) lacking endogenous PDZ domain ligands can be engineered as fusion products containing C-terminal PDZ domain ligand peptides or internal, N- or C-terminal PDZ domains and then can be purified by the same method. Using this method, we recovered recombinant GFP fused to a PDZ domain ligand in active form as verified by fluorescence yield. Similarly, chloramphenicol acetyltransferase (CAT) and β-Galactosidase (LacZ) fused to a C-terminal PDZ domain ligand or an N-terminal PDZ domain were purified in active form as assessed by enzymatic assay. In general, PDZ domains and ligands derived from PSD-95 were superior to those from nNOS for this method. PDZ Domain Affinity Chromatography promises to be a versatile and effective method for purification of a wide variety of natural and recombinant proteins. Copyright © 2014 Elsevier Inc. All rights reserved.

NADH fluorescence lifetime analysis of the effect of magnesium ions on ALDH2

USDA-ARS?s Scientific Manuscript database

ALDH2 catalyzes oxidation of toxic aldehydes to their corresponding carboxylic acids. Magnesium ions influence enzyme activity in part by increasing NADH binding affinity. Traditional fluorescence measurements have monitored the blue shift of the NADH fluorescence spectrum to elucidate the extent of...

NADH fluorescence lifetime analysis of the effect of magnesium ions on ALDH2

USDA-ARS?s Scientific Manuscript database

Aldehyde dehydrogenase 2 (ALDH2) catalyzes oxidation of toxic aldehydes to carboxylic acids. Physiologic levels of Mg2+ ions influence enzyme activity in part by increasing NADH binding affinity. Traditional fluorescence measurements monitor the blue shift of the NADH fluorescence spectrum to study ...

Evaluation of chemical fluorescent dyes as a protein conjugation partner for live cell imaging.

PubMed

Hayashi-Takanaka, Yoko; Stasevich, Timothy J; Kurumizaka, Hitoshi; Nozaki, Naohito; Kimura, Hiroshi

2014-01-01

To optimize live cell fluorescence imaging, the choice of fluorescent substrate is a critical factor. Although genetically encoded fluorescent proteins have been used widely, chemical fluorescent dyes are still useful when conjugated to proteins or ligands. However, little information is available for the suitability of different fluorescent dyes for live imaging. We here systematically analyzed the property of a number of commercial fluorescent dyes when conjugated with antigen-binding (Fab) fragments directed against specific histone modifications, in particular, phosphorylated H3S28 (H3S28ph) and acetylated H3K9 (H3K9ac). These Fab fragments were conjugated with a fluorescent dye and loaded into living HeLa cells. H3S28ph-specific Fab fragments were expected to be enriched in condensed chromosomes, as H3S28 is phosphorylated during mitosis. However, the degree of Fab fragment enrichment on mitotic chromosomes varied depending on the conjugated dye. In general, green fluorescent dyes showed higher enrichment, compared to red and far-red fluorescent dyes, even when dye:protein conjugation ratios were similar. These differences are partly explained by an altered affinity of Fab fragment after dye-conjugation; some dyes have less effect on the affinity, while others can affect it more. Moreover, red and far-red fluorescent dyes tended to form aggregates in the cytoplasm. Similar results were observed when H3K9ac-specific Fab fragments were used, suggesting that the properties of each dye affect different Fab fragments similarly. According to our analysis, conjugation with green fluorescent dyes, like Alexa Fluor 488 and Dylight 488, has the least effect on Fab affinity and is the best for live cell imaging, although these dyes are less photostable than red fluorescent dyes. When multicolor imaging is required, we recommend the following dye combinations for optimal results: Alexa Fluor 488 (green), Cy3 (red), and Cy5 or CF640 (far-red).

Exploring high-affinity binding properties of octamer peptides by principal component analysis of tetramer peptides.

PubMed

Kume, Akiko; Kawai, Shun; Kato, Ryuji; Iwata, Shinmei; Shimizu, Kazunori; Honda, Hiroyuki

2017-02-01

To investigate the binding properties of a peptide sequence, we conducted principal component analysis (PCA) of the physicochemical features of a tetramer peptide library comprised of 512 peptides, and the variables were reduced to two principal components. We selected IL-2 and IgG as model proteins and the binding affinity to these proteins was assayed using the 512 peptides mentioned above. PCA of binding affinity data showed that 16 and 18 variables were suitable for localizing IL-2 and IgG high-affinity binding peptides, respectively, into a restricted region of the PCA plot. We then investigated whether the binding affinity of octamer peptide libraries could be predicted using the identified region in the tetramer PCA. The results show that octamer high-affinity binding peptides were also concentrated in the tetramer high-affinity binding region of both IL-2 and IgG. The average fluorescence intensity of high-affinity binding peptides was 3.3- and 2.1-fold higher than that of low-affinity binding peptides for IL-2 and IgG, respectively. We conclude that PCA may be used to identify octamer peptides with high- or low-affinity binding properties from data from a tetramer peptide library. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Zinc(II) binds to the neuroprotective peptide humanin.

PubMed

Armas, Ambar; Sonois, Vanessa; Mothes, Emmanuelle; Mazarguil, Honoré; Faller, Peter

2006-10-01

The abnormal accumulation of the peptide amyloid-beta in the form of senile (or amyloid) plaques is one of the hallmarks of Alzheimer's disease (AD). Zinc ions have been implicated in AD and plaques formation. Recently, the peptide humanin has been discovered. Humanin showed neuroprotective activity against amyloid-beta insults. Here the question investigated is if humanin could interact directly with Zn(II). It is shown that Zn(II) and its substitutes Cd(II)/Co(II) bind to humanin via a thiolate bond from the side chain of the single cysteine at position 8. The low intensity of the d-d bands of Co(II)-humanin indicated an octahedral coordination geometry. Titration experiments suggest that Zn(II) binds to humanin with an apparent affinity in the low muM range. This apparent Zn-binding affinity is in the same order as for amyloid-beta and glutathione and could thus be of physiological relevance.

A Fluorescence Polarization Biophysical Assay for the Naegleria DNA Hydroxylase Tet1.

PubMed

Marholz, Laura J; Wang, Wei; Zheng, Yu; Wang, Xiang

2016-02-11

The discovery of the 5-methylcytosine (5mC) oxidation by the ten-eleven translocation (Tet) protein family was an important advancement in our understanding of DNA-modified epigenetics. Potent inhibitors of these proteins are greatly desired for both the understanding of the functions of these enzymes and to serve as eventual therapeutic leads. So far, the discovery of such small molecules with high affinity has been quite limited. Original tools to screen for activity are greatly needed in order to accelerate this process. Here we present a novel fluorescent probe, and the results of a fluorescence polarization-based binding assay for Naegleria Tet1, a homologue to mammalian Tet. A fluorescence polarization-based competition assay was also established and applied to the rapid and quantitative measurement of the binding affinity of the cofactor αKG and several known Tet1 inhibitors.

Novel Zn(II) complexes of 1,3-diphenyl-4-(arylazo)pyrazol-5-one derivatives: Synthesis, spectroscopic properties, DFT calculations and first order nonlinear optical properties

NASA Astrophysics Data System (ADS)

Abdel-Latif, Samir A.; Mohamed, Adel A.

2018-03-01

Eight novel Zn(II) complexes with substituted 1,3-diphenyl-4-(arylazo)pyrazol-5-one (L1-L4) derivatives have been synthesized and elucidated using various physicochemical techniques. Quantum mechanical calculations of energies, geometries were done by DFT using B3LYP/GEN functional combined with 6.311G (d,p) and LAN2DZ basis sets. The analyses of HOMO and LUMO have been used to explain the charge transfer within the ligands and complexes. The calculated small energy gap between HOMO and LUMO energies shows that the charge transfer occurs within Zn(II) complexes. Geometrical parameters, molecular electrostatic potential maps (MEP) and total electron densities analyses of the ligands and their Zn complexes have been carried out. Molecular stability, hyperconjugative interactions, intramolecular charge transfer (ICT) and bond strength has been investigated by the applying of natural bond orbital (NBO) analysis. Total static dipole moment (μ), the mean polarizability (<α>), the anisotropy of the polarizability (Δα), the mean first-order hyperpolarizability (<β>) have been also performed. The obtained values show that Zn(II) complexes is brilliant candidate to NLO materials. The analyses of the 1:1 complexes indicate that the Zn(II) ion is five-coordinated with water molecules at axial position in case of L1, L2 and L4 whereas, six-coordinated with L3 and non-electrolytic behaviour of complexes indicates the absence of counter ion.

Electrochemical affinity biosensors for detection of mycotoxins: A review.

PubMed

Vidal, Juan C; Bonel, Laura; Ezquerra, Alba; Hernández, Susana; Bertolín, Juan R; Cubel, Carlota; Castillo, Juan R

2013-11-15

This review discusses the current state of electrochemical biosensors in the determination of mycotoxins in foods. Mycotoxins are highly toxic secondary metabolites produced by molds. The acute toxicity of these results in serious human and animal health problems, although it has been only since early 1960s when the first studied aflatoxins were found to be carcinogenic. Mycotoxins affect a broad range of agricultural products, most important cereals and cereal-based foods. A majority of countries, mentioning especially the European Union, have established preventive programs to control contamination and strict laws of the permitted levels in foods. Official methods of analysis of mycotoxins normally requires sophisticated instrumentation, e.g. liquid chromatography with fluorescence or mass detectors, combined with extraction procedures for sample preparation. For about sixteen years, the use of simpler and faster analytical procedures based on affinity biosensors has emerged in scientific literature as a very promising alternative, particularly electrochemical (i.e., amperometric, impedance, potentiometric or conductimetric) affinity biosensors due to their simplicity and sensitivity. Typically, electrochemical biosensors for mycotoxins use specific antibodies or aptamers as affinity ligands, although recombinant antibodies, artificial receptors and molecular imprinted polymers show potential utility. This article deals with recent advances in electrochemical affinity biosensors for mycotoxins and covers complete literature from the first reports about sixteen years ago. Copyright © 2013 Elsevier B.V. All rights reserved.

Evaluation of homologous, heterologous, and affinity conjugates for the serodiagnosis of Toxoplasma gondii and Neospora caninum in maned wolves (Chrysocyon brachyurus).

PubMed

Silva, D A O; Vitaliano, S N; Mineo, T W P; Ferreira, R A; Bevilacqua, E; Mineo, J R

2005-10-01

Use of serological tests in the diagnosis of infectious diseases in wild animals has several limitations, primarily the difficulty of obtaining species-specific reagents. Wild canids, such as maned wolves (Chrysocyon brachyurus), are highly predisposed to infection by Toxoplasma gondii and, to a lesser extent, to Neospora caninum. The aim of the present study was to evaluate homologous, heterologous, and affinity conjugates in enzyme-linked immunosorbent assays (ELISAs) and indirect fluorescent antibody tests (IFATs) for detecting immunoglobulin (Ig) G antibodies against T. gondii and N. caninum in maned wolves. Serum samples were obtained from 59 captive animals in Brazil and tested by ELISA for T. gondii serology and IFAT for N. caninum serology using 3 different enzymatic and fluorescent conjugates: homologous (guinea pig anti-maned wolf IgG-peroxidase and -fluorescein isothiocyanate [FITC]), heterologous (rabbit anti-dog IgG-peroxidase and -FITC), and affinity (protein A-peroxidase and -FITC). Seropositivity to T. gondii was comparable among the homologous (69.5%), heterologous (74.6%), and affinity (71.2%) enzymatic conjugates. A significant positive correlation was found between the antibody levels determined by the 3 enzymatic conjugates. The highest mean antibody levels (ELISA index = 4.5) were observed with the protein A-peroxidase conjugate. The same seropositivity to N. caninum (8.5%) was found with the homologous and heterologous fluorescent conjugates, but protein A-FITC was not able to detect or confirm any positive samples with homologous or heterologous conjugates. Our results demonstrate that homologous, heterologous, and affinity conjugates might be used in ELISA for serological assays of T. gondii in wild canids, whereas for N. caninum infection, only the homologous or heterologous fluorescent conjugates have been shown to be useful.

Comparative absorption spectroscopy involving 4f-4f transitions to explore the kinetics of simultaneous coordination of uracil with Nd(III) and Zn(II) and its associated thermodynamics

NASA Astrophysics Data System (ADS)

Victory Devi, Ch.; Rajmuhon Singh, N.

2011-10-01

The interaction of uracil with Nd(III) has been explored in presence and absence of Zn(II) using the comparative absorption spectroscopy involving the 4f-4f transitions in different solvents. The complexation of uracil with Nd(III) is indicated by the change in intensity of 4f-4f bands expressing in terms of significant change in oscillator strength and Judd-Ofelt parameters. Intensification of this bands became more prominent in presence of Zn(II) suggesting the stimulative effect of Zn(II) towards the complexation of Nd(III) with uracil. Other spectral parameters namely Slator-Condon ( Fk's), nephelauxetic effect ( β), bonding ( b1/2) and percent covalency ( δ) parameters are computed to correlate their simultaneous binding of metal ions with uracil. The sensitivities of the observed 4f-4f transitions towards the minor coordination changes around Nd(III) has been used to monitor the simultaneous coordination of uracil with Nd(III) and Zn(II). The variation of intensities (oscillator strengths and Judd-Ofelt parameters) of 4f-4f bands during the complexation has helped in following the heterobimetallic complexation of uracil. Rate of complexation with respect to hypersensitive transition was evaluated. Energy of activation and thermodynamic parameters for the complexation reaction were also determined.

A red-emitting indolium fluorescence probe for membranes - flavonoids interactions.

PubMed

Gao, Qingyun; Liu, Han; Ding, Qiongjie; Du, Jinya; Liu, Chunlin; Yang, Wei; Shen, Ping; Yang, Changying

2018-05-01

The red-emitting indolium derivative compound (E)-2-(4-(diphenylamino)styryl)-1,3,3-trimethyl-3H-indol-1-ium iodide (H3) was demonstrated as a sensitive membrane fluorescence probe. The probe located at the interface of liposomes when mixed showed much fluorescence enhancement by inhibiting the twisted intramolecular charge transfer state. After ultrasonic treatment, it penetrated into lipid bilayers with the emissions leveling off and a rather large encapsulation efficiency (71.4%) in liposomes. The ζ-potential and particle size measurement confirmed that the charged indolium group was embedded deeply into lipid bilayers. The probe was then used to monitor the affinities of antioxidant flavonoids for membranes. It was verified that quercetin easily interacted with liposomes and dissociated the probe from the internal lipid within 60 s under the condition of simply mixing. The assessment of binding affinities of six flavonoids and the coincident results with their antioxidation activities indicated that it was a promising membrane probe for the study of drug bio-affinities. Copyright © 2018 John Wiley & Sons, Ltd.

The effect of ligand affinity on integrins' lateral diffusion in cultured cells.

PubMed

Mainali, Dipak; Smith, Emily A

2013-04-01

The role of ligand affinity in altering αPS2CβPS integrins' lateral mobility was studied using single particle tracking (SPT) with ligand-functionalized quantum dots (QDs) and fluorescence recovery after photobleaching (FRAP) with fluorescent protein tagged integrins. Integrins are ubiquitous transmembrane proteins that are vital for numerous cellular functions, including bidirectional signaling and cell anchorage. Wild-type and high ligand affinity mutant (αPS2CβPS-V409D) integrins were studied in S2 cells. As measured by SPT, the integrin mobile fraction decreased by 22% and had a 4× slower diffusion coefficient for αPS2CβPS-V409D compared to wild-type integrins. These differences are partially the result of αPS2CβPS-V409D integrins' increased clustering. For the wild-type integrins, the average of all diffusion coefficients measured by SPT was statistically similar to the ensemble FRAP results. A 75% slower average diffusion coefficient was measured by SPT compared to FRAP for αPS2CβPS-V409D integrins, and this may be the result of SPT measuring only ligand-bound integrins, in contrast all ligand-bound and ligand-unbound integrins are averaged in FRAP measurements. Specific binding of the ligand-functionalized QDs was 99% for integrin expressing cells. The results prove that the ligand binding affinity affects the lateral dynamics of a subset of integrins based on the complementary SPT and FRAP data.

Method and apparatus for detection of fluorescently labeled materials

DOEpatents

Stern, David; Fiekowsky, Peter

2004-05-25

Fluorescently marked targets bind to a substrate 230 synthesized with polymer sequences at known locations. The targets are detected by exposing selected regions of the substrate 230 to light from a light source 100 and detecting the photons from the light fluoresced therefrom, and repeating the steps of exposure and detection until the substrate 230 is completely examined. The resulting data can be used to determine binding affinity of the targets to specific polymer sequences.

Competitive adsorption of Pb(II), Cu(II), and Zn(II) ions onto hydroxyapatite-biochar nanocomposite in aqueous solutions

NASA Astrophysics Data System (ADS)

Wang, Yu-Ying; Liu, Yu-Xue; Lu, Hao-Hao; Yang, Rui-Qin; Yang, Sheng-Mao

2018-05-01

A hydroxyapatite-biochar nanocomposite (HAP-BC) was successfully fabricated and its physicochemical properties characterized. The analyses showed that HAP nanoparticles were successfully loaded on the biochar surface. The adsorption of Pb(II), Cu(II), and Zn(II) by HAP-BC was systematically studied in single and ternary metal systems. The results demonstrated that pH affects the adsorption of heavy metals onto HAP-BC. Regarding the adsorption kinetics, the pseudo-second-order model showed the best fit for all three heavy metal ions on HAP-BC. In both single and ternary metal ion systems, the adsorption isotherm of Pb(II) by HAP-BC followed Langmuir model, while those of Cu(II) and Zn(II) fitted well with Freundlich model. The maximum adsorption capacity for each tested metal by HAP-BC was higher than that of pristine rice straw biochar (especially for Pb(II)) or those of other reported adsorbents. Therefore, HAP-BC could explore as a new material for future application in heavy metal removal.

Application of silica fume as a new SP-extractor for trace determination of Zn(II) and Cd(II) in pharmaceutical and environmental samples by square-wave anodic stripping voltammetry

NASA Astrophysics Data System (ADS)

Ahmed, Salwa A.; Gaber, Ahmed A. Abdel; Rahim, Asmaa M. Abdel

2017-05-01

In this work, silica fume (SF) is used as a solid-phase extractor for extraction of Zn(II) and Cd(II) from aqueous solutions. Characterization of SF is performed by Fourier transform infrared, X-ray diffraction, transmission and scanning electron microscopy. The optimum experimental conditions for the two metal ions are investigated using batch and column techniques. The maximum adsorption capacity values are found to be 54.13 and 121.28 mg g-1 at the optimum pH 6.0 and 8.0 for Zn(II) and Cd(II), respectively. The equilibrium data are analyzed using the Langmuir, Freundlich, and Temkin isotherms by nonlinear regression analysis. Also, the kinetics analysis revealed that the overall adsorption process is successfully fitted with the pseudo-second-order model. The method is applied for determination of the target metal ions in pharmaceutical and environmental samples using square-wave anodic stripping voltammetry. The limit of detection (LOD) values are 0.102 and 1.43 × 10-3 mg L-1 for Zn(II) and Cd(II), respectively. The percentage recovery values are 98.8-100.5 % which indicate the success of the proposed method for determination of Zn(II) and Cd(II) without interfering effects.

Synthesis, characterization, antimicrobial activity and DFT studies of 2-(pyrimidin-2-ylamino)naphthalene-1,4-dione and its Mn(II), Co(II), Ni(II) and Zn(II) complexes

NASA Astrophysics Data System (ADS)

Chioma, Festus; Ekennia, Anthony C.; Ibeji, Collins U.; Okafor, Sunday N.; Onwudiwe, Damian C.; Osowole, Aderoju A.; Ujam, Oguejiofo T.

2018-07-01

A pyrimidine-based ligand, 2-(pyrimidin-2-ylamino)naphthalene-1,4-dione (L), has been synthesized by the reaction of 2-aminopyrimidine with 2-hydroxy-1,4-napthoquinone. Reaction of the ligand with Ni(II), Co(II), Mn(II) and Zn(II) acetate gave the corresponding metal complexes which were characterized by spectroscopic techniques, (infrared, electronic), elemental analysis, room-temperature magnetometry, conductance measurements and thermogravimetry-differential scanning calorimetry (TG-DSC) analyses. The room-temperature magnetic data and electronic spectral measurements of the complexes gave evidence of 4-coordinate square planar/tetrahedral geometry. The thermal analyses values obtained indicated the monohydrate complexes. The antimicrobial screening of the compounds showed mild to very good results. The Mn(II) complex showed the best result within in the range of 11.5-29 mm. The electronic, structural and spectroscopic properties of the complexes were further discussed using density functional theory. Molecular docking studies showed significant binding affinity with the drug targets and the metal complexes have potentials to be used as drugs.

Synthesis, spectroscopic characterization and biological activities of N4O2 Schiff base ligand and its metal complexes of Co(II), Ni(II), Cu(II) and Zn(II)

NASA Astrophysics Data System (ADS)

Al-Resayes, Saud I.; Shakir, Mohammad; Abbasi, Ambreen; Amin, Kr. Mohammad Yusuf; Lateef, Abdul

The Schiff base ligand, bis(indoline-2-one)triethylenetetramine (L) obtained from condensation of triethylenetetramine and isatin was used to synthesize the complexes of type, [ML]Cl2 [M = Co(II), Ni(II), Cu(II) and Zn(II)]. L was characterized on the basis of the results of elemental analysis, FT-IR, 1H and 13C NMR, mass spectroscopic studies. The stoichiometry, bonding and stereochemistries of complexes were ascertained on the basis of results of elemental analysis, magnetic susceptibility values, molar conductance and various spectroscopic studies. EPR, UV-vis and magnetic moments revealed an octahedral geometry for complexes. L and its Cu(II) and Zn(II) complexes were screened for their antibacterial activity. Analgesic activity of Cu(II) and Zn(II) complexes was also tested in rats by tail flick method. Both complexes were found to possess good antibacterial and moderate analgesic activity.

Synthesis, characterization of 1,2,4-triazole Schiff base derived 3d-metal complexes: Induces cytotoxicity in HepG2, MCF-7 cell line, BSA binding fluorescence and DFT study

NASA Astrophysics Data System (ADS)

Tyagi, Prateek; Tyagi, Monika; Agrawal, Swati; Chandra, Sulekh; Ojha, Himanshu; Pathak, Mallika

2017-01-01

Two novel Schiff base ligands H2L1 and H2L2 have been synthesized by condensation reaction of amine derivative of 1,2,4-triazole moiety with 2-hydroxy-4-methoxybenzaldehyde. Co(II), Ni(II), Cu(II) and Zn(II) of the synthesized Schiff bases were prepared by using a molar ratio of ligand:metal as 1:1. The structure of the Schiff bases and synthesized metal complexes were established by 1H NMR, UV-Vis, IR, Mass spectrometry and molar conductivity. The thermal stability of the complexes was study by TGA. Fluorescence quenching mechanism of metal complexes 1-4 show that Zn(II) and Cu(II) complex binds more strongly to BSA. In DFT studies the geometries of Schiff bases and metal complexes were fully optimized with respect to the energy using the 6-31 + g(d,p) basis set. The spectral data shows that the ligands behaves as binegative tridentate. On the basis of the spectral studies, TGA and DFT data an octahedral geometry has been assigned for Co(II), Ni(II), square planar for Cu(II) and tetrahedral for Zn(II) complexes. The anticancer activity were screened against human breast cancer cell line (MCF-7) and human hepatocellular liver carcinoma cell line (Hep-G2). Result indicates that metal complexes shows increase cytotoxicity in proliferation to cell lines as compared to free ligand.

Fluorescent vancomycin and terephthalate comodified europium-doped layered double hydroxides nanoparticles: synthesis and application for bacteria labelling

NASA Astrophysics Data System (ADS)

Sun, Jianchao; Fan, Hai; Wang, Nan; Ai, Shiyun

2014-09-01

Vancomycin (Van)- and terephthalate (TA)-comodified europium-doped layered double hydroxides (Van-TA-Eu-LDHs) nanoparticles were successfully prepared by a two-step method, in which, TA acted as a sensitizer to enhance the fluorescent property and Van was modified on the surface of LDH to act as an affinity reagent to bacteria. The obtained products were characterized by X-ray diffraction, transmission electron microscope and fluorescent spectroscopy. The results demonstrated that the prepared Van- and TA-comodified europium-doped layered double hydroxides (Van-TA-Eu-LDHs) nanoparticles with diameter of 50 nm in size showed highly efficient fluorescent property. Furthermore, due to the high affinity of Van to bacteria, the prepared Van-TA-Eu-LDHs nanoparticles showed efficient bacteria labelling by fluorescent property. The prepared nanoparticles may have wide applications in the biological fields, such as biomolecular labelling and cell imaging.

Luminescent sensing of Cu2+, CrO42- and photocatalytic degradation of methyl violet by Zn(II) metal-organic framework (MOF) having 5,5‧-(1H-2,3,5-triazole-1,4-diyl)diisophthalic acid ligand

NASA Astrophysics Data System (ADS)

Wu, Weiping; Li, Baohong; Gu, Chuying; Wang, Jun; Singh, Amita; Kumar, Abhinav

2017-11-01

A porous Zn(II) metal-organic framework (MOF) [Zn(H2L)(4,4‧-bipy)0.5]n (1) has been selected and its luminescence sensing for cations and anions as well as the photocatalytic property against methyl violet have been explored. Luminescence studies indicated that 1 could be an efficient multifunctional fluorescent material for highly sensitive detection of metal cation Cu2+ and anions CrO42-. The luminescence intensity of 1 was found to decrease proportionately with increase in the concentration of Cu2+ and CrO42-. Furthermore, the photocatalytic property of 1 for degradation of the methyl violet (MV) have been explored and a possible photocatalytic mechanism have been proposed using density of states (DOS) and partial DOS (pDOS) calculations.

Probing the nucleotide binding domain of the osmoregulator EnvZ using fluorescent nucleotide derivatives.

PubMed

Plesniak, Leigh; Horiuchi, Yuki; Sem, Daniel; Meinenger, David; Stiles, Linda; Shaffer, Jennifer; Jennings, Patricia A; Adams, Joseph A

2002-11-26

EnvZ is a histidine protein kinase important for osmoregulation in bacteria. While structural data are available for this enzyme, the nucleotide binding pocket is not well characterized. The ATP binding domain (EnvZB) was expressed, and its ability to bind nucleotide derivatives was assessed using equilbrium and stopped-flow fluorescence spectroscopy. The fluorescence emission of the trinitrophenyl derivatives, TNP-ATP and TNP-ADP, increase upon binding to EnvZB. The fluorescence enhancements were quantitatively abolished in the presence of excess ADP, indicating that the fluorescent probes occupy the nucleotide binding pocket. Both TNP-ATP and TNP-ADP bind to EnvZB with high affinity (K(d) = 2-3 microM). The TNP moiety attached to the ribose ring does not impede access of the fluorescent nucleotide into the binding pocket. The association rate constant for TNP-ADP is 7 microM(-1) s(-1), a value consistent with those for natural nucleotides and the eucaryotic protein kinases. Using competition experiments, it was found that ATP and ADP bind 30- and 150-fold more poorly, respectively, than the corresponding TNP-derivatized forms. Surprisingly, the physiological metal Mg(2+) is not required for ADP binding and only enhances ATP affinity by 3-fold. Although portions of the nucleotide pocket are disordered, the recombinant enzyme is highly stable, unfolding only at temperatures in excess of 70 degrees C. The unusually high affinity of the TNP derivatives compared to the natural nucleotides suggests that hydrophobic substitutions on the ribose ring enforce an altered binding mode that may be exploited for drug design strategies.

Evaluation of affinity and pseudo-affinity adsorption processes for penicillin acylase purification.

PubMed

Fonseca, L P; Cabral, J M

1996-01-01

Affinity ligand (6-Aminopenicillanic acid, Amoxycillin, Ampicillin, Benzylpenicillin and 4-Phenylbutylanzine) of penicillin acylase (EC 3.5.1.11) were attached to hydrophilic gels like Sepharose 4B-CNBr and Minileak 'medium'. Ampicillin and 4-Phenylbutylamine were the affinity ligands that presented the higher concentrations attached to both gels. Penicillin acylase adsorption on these affinity gels was mainly dependent on the activated group of the gel, the affinity ligand attached and the experimental conditions of enzyme adsorption. Under affinity conditions only the ligands Amoxycillin, Ampicillin and 4-Phenylbutylamine, immobilized on Minileak, adsorbed the enzyme from osmotic shock extracts at different pH values. These affinity ligand systems were characterized by low adsorption capacities of penicillin acylase activity (1.2-2.1 IU mL-1 gel) and specific activity (1.5-2.9 IU mg-1 prot). Under pseudo-affinity conditions all the ligands attached both activated to gels (Sepharose 4B-CNBr and Minileak) adsorbed the enzyme. The affinity gels were characterized by higher values of adsorption capacity (3.7 and 55.6 IU mL-1 gel) and adsorbed specific activity (2.0 and 6.1 IU mg-1 prot) than those observed under affinity conditions. The space arm of Minileak gel, shown to be fundamental to enzyme adsorption under affinity conditions, preferentially adsorbed proteins in relation to the enzyme under pseudo-affinity conditions. However, this effect was partially minimized when the gel was derivatized by the affinity ligands at concentrations higher than 6 mumol mL-1 gel. Ampicillin was the affinity ligand that presented the best results for specific adsorption of penicillin acylase under affinity and pseudo-affinity adsorption processes. The Sepharose 4B-CNBr derivatized gel also presented a good adsorption capacity of enzyme activity (26.8 IU mL-1 gel) under pseudo-affinity adsorption processes.

Fluorescent nanoscale zinc(II)-carboxylate coordination polymers for explosive sensing.

PubMed

Zhang, Chengyi; Che, Yanke; Zhang, Zengxing; Yang, Xiaomei; Zang, Ling

2011-02-28

Fluorescent nanoscale coordination polymers with cubic morphology and long range ordered structure were fabricated and exhibited efficient sensing for both nitroaromatic explosive and nitromethane due to large surface area to volume ratio and strong binding affinity to explosive molecules.

A Comparative Study of the Application of Fluorescence Excitation-Emission Matrices Combined with Parallel Factor Analysis and Nonnegative Matrix Factorization in the Analysis of Zn Complexation by Humic Acids

PubMed Central

Boguta, Patrycja; Pieczywek, Piotr M.; Sokołowska, Zofia

2016-01-01

The main aim of this study was the application of excitation-emission fluorescence matrices (EEMs) combined with two decomposition methods: parallel factor analysis (PARAFAC) and nonnegative matrix factorization (NMF) to study the interaction mechanisms between humic acids (HAs) and Zn(II) over a wide concentration range (0–50 mg·dm−3). The influence of HA properties on Zn(II) complexation was also investigated. Stability constants, quenching degree and complexation capacity were estimated for binding sites found in raw EEM, EEM-PARAFAC and EEM-NMF data using mathematical models. A combination of EEM fluorescence analysis with one of the proposed decomposition methods enabled separation of overlapping binding sites and yielded more accurate calculations of the binding parameters. PARAFAC and NMF processing allowed finding binding sites invisible in a few raw EEM datasets as well as finding totally new maxima attributed to structures of the lowest humification. Decomposed data showed an increase in Zn complexation with an increase in humification, aromaticity and molecular weight of HAs. EEM-PARAFAC analysis also revealed that the most stable compounds were formed by structures containing the highest amounts of nitrogen. The content of oxygen-functional groups did not influence the binding parameters, mainly due to fact of higher competition of metal cation with protons. EEM spectra coupled with NMF and especially PARAFAC processing gave more adequate assessments of interactions as compared to raw EEM data and should be especially recommended for modeling of complexation processes where the fluorescence intensities (FI) changes are weak or where the processes are interfered with by the presence of other fluorophores. PMID:27782078

Quantum dot immunoassays in renewable surface column and 96-well plate formats for the fluorescence detection of Botulinum neurotoxin using high-affinity antibodies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Warner, Marvin G.; Grate, Jay W.; Tyler, Abby J.

2009-09-01

A fluorescence sandwich immunoassay using high affinity antibodies and quantum dot (QD) reporters has been developed for detection of botulinum toxin serotype A (BoNT/A). For the development of the assay, a nontoxic recombinant fragment of the holotoxin (BoNT/A-HC-fragment) has been used as a structurally valid simulant for the full toxin molecule. The antibodies used, AR4 and RAZ1, bind to nonoverlapping epitopes present on both the full toxin and on the recombinant fragment. In one format, the immunoassay is carried out in a 96-well plate with detection in a standard plate reader. Detection down to 31 pM of the BoNT/Hc-fragment wasmore » demonstrated with a total incubation time of 3 hours, using AR4 as the capture antibody and QD-coupled RAZ1 as the reporter. In a second format, the AR4 capture antibody was coupled to Sepharose beads, and the immunochemical reactions were carried out in microcentrifuge tubes with an incubation time of 1 hour. These beads were subsequently captured and concentrated in a rotating rod “renewable surface” flow cell as part of a sequential injection fluidic system. This flow cell was equipped with a fiber optic system for fluorescence measurements. In PBS buffer solution matrix, the BoNT/A-HC-fragment was detected to concentrations as low as 5 pM using the fluidic measurement approach.« less

A Class of Multiresponsive Colorimetric and Fluorescent pH Probes via Three Different Reaction Mechanisms of Salen Complexes: A Selective and Accurate pH Measurement.

PubMed

Cheng, Jinghui; Gou, Fei; Zhang, Xiaohong; Shen, Guangyu; Zhou, Xiangge; Xiang, Haifeng

2016-09-19

We report a class of multiresponsive colorimetric and fluorescent pH probes based on three different reaction mechanisms including cation exchange, protonation, and hydrolysis reaction of K(I), Ca(II), Zn(II), Cu(II), Al(III), and Pd(II) Salen complexes. Compared with traditional pure organic pH probes, these complex-based pH probes exhibited a much better selectivity due to the shielding function of the filled-in metal ion in the complex. Their pH sensing performances were affected by the ligand structure and the central metal ion. This work is the first report of "off-on-on'-off" colorimetric and fluorescent pH probes that possess three different reaction mechanisms and should inspire the design of multiple-responsive probes for important analytes in biological systems.

Introducing a fluorescence-based standard to quantify protein partitioning into membranes.

PubMed

Thomas, Franziska A; Visco, Ilaria; Petrášek, Zdeněk; Heinemann, Fabian; Schwille, Petra

2015-11-01

The affinity of peripheral membrane proteins for a lipid bilayer can be described using the partition coefficient (KP). Although several methods to determine KP are known, all possess limitations. To address some of these issues, we developed both: a versatile method based on single molecule detection and fluorescence imaging for determining KP, and a simple measurement standard employing hexahistidine-tagged enhanced green fluorescent protein (eGFP-His6) and free standing membranes of giant unilamellar vesicles (GUVs) functionalized with NTA(Ni) lipids as binding sites. To ensure intrinsic control, our method features two measurement modes. In the single molecule mode, fluorescence correlation spectroscopy (FCS) is applied to quantify free and membrane associated protein concentrations at equilibrium and calculate KP. In the imaging mode, confocal fluorescence images of GUVs are recorded and analyzed with semi-automated software to extract protein mean concentrations used to derive KP. Both modes were compared by determining the affinity of our standard, resulting in equivalent KP values. As observed in other systems, eGFP-His6 affinity for membranes containing increasing amounts of NTA(Ni) lipids rises in a stronger-than-linear fashion. We compared our dual approach with a FCS-based assay that uses large unilamellar vesicles (LUVs), which however fails to capture the stronger-than-linear trend for our NTA(Ni)-His6 standard. Hence, we determined the KP of the MARCKS effector domain with our FCS approach on GUVs, whose results are consistent with previously published data using LUVs. We finally provide a practical manual on how to measure KP and understand it in terms of molecules per lipid surface. Copyright © 2015. Published by Elsevier B.V.

Identification of lanthanum-specific peptides for future recycling of rare earth elements from compact fluorescent lamps.

PubMed

Lederer, Franziska L; Curtis, Susan B; Bachmann, Stefanie; Dunbar, W Scott; MacGillivray, Ross T A

2017-05-01

As components of electronic scrap, rare earth minerals are an interesting but little used source of raw materials that are highly important for the recycling industry. Currently, there exists no cost-efficient technology to separate rare earth minerals from an electronic scrap mixture. In this study, phage surface display has been used as a key method to develop peptides with high specificity for particular inorganic targets in electronic scrap. Lanthanum phosphate doped with cerium and terbium as part of the fluorescent phosphors of spent compact fluorescent lamps (CFL) was used as a target material of economic interest to test the suitability of the phage display method to the separation of rare earth minerals. One random pVIII phage library was screened for peptide sequences that bind specifically to the fluorescent phosphor LaPO 4 :Ce 3+ ,Tb 3+ (LAP). The library contained at least 100 binding pVIII peptides per phage particle with a diversity of 1 × 10 9 different phage per library. After three rounds of enrichment, a phage clone containing the surface peptide loop RCQYPLCS was found to bind specifically to LAP. Specificity and affinity of the identified phage bound peptide was confirmed by using binding and competition assays, immunofluorescence assays, and zeta potential measurements. Binding and immunofluorescence assays identified the peptide's affinity for the fluorescent phosphor components CAT (CeMgAl 11 O 19 :Tb 3+ ) and BAM (BaMgAl 10 O 17 :Eu 2+ ). No affinity was found for other fluorescent phosphor components such as YOX (Y 2 O 3 :Eu 3+ ). The binding specificity of the RCQYPLCS peptide loop was improved 3-51-fold by using alanine scanning mutagenesis. The identification of peptides with high specificity and affinity for special components in the fluorescent phosphor in CFLs provides a potentially new strategic approach to rare earth recycling. Biotechnol. Bioeng. 2017;114: 1016-1024. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals

Reduction of high-affinity beta2-adrenergic receptor binding by hyperforin and hyperoside on rat C6 glioblastoma cells measured by fluorescence correlation spectroscopy.

PubMed

Prenner, Lars; Sieben, Anne; Zeller, Karin; Weiser, Dieter; Häberlein, Hanns

2007-05-01

Beta-adrenergic receptors (beta-AR) are potential targets for antidepressants. Desensitization and downregulation of beta-AR are discussed as possible modes of action for antidepressants. We have investigated the effects of hyperforin and hyperoside, compounds with potentially antidepressant activity from St. John's Wort, on the binding behavior and dynamics of beta2-AR in living rat C6 glioblastoma cells, compared to desipramine (desmethylimipramine; DMI) by means of fluorescence correlation spectroscopy (FCS) and fluorescence microscopy. FCS-binding studies with the fluorescently labeled ligand Alexa532-noradrenaline (Alexa532-NA) binding to beta2-AR of C6 cells showed a significant reduction in total beta2-AR binding after preincubation with hyperforin and hyperoside for 3 days, respectively, which was also found for DMI. This was mainly observed in high-affinity receptor-ligand complexes with hindered lateral mobility (D2 = 1.1 (+/-0.4) microm2/s) in the biomembrane. However, internalization of beta2-AR was found neither in z-scans of these C6 cells nor in HEK 293 cells stably transfected with GFP-tagged beta2-adrenergic receptors (beta2AR-GFP) after incubation up to 6 days with either DMI, hyperforin, or hyperoside. Thus, under these conditions reduction of beta2-AR binding was not mediated by receptor internalization. Additionally, preincubation of C6 cells with DMI, hyperforin, and hyperoside led to a loss of second messenger cAMP after beta2-adrenergic stimulating conditions with terbutaline. Our current results indicate that hyperforin and hyperoside from St. John's Wort, as well as DMI, reduce beta2-adrenergic sensitivity in C6 cells, emphasizing the potential usefulness of St. John's Wort dry extracts in clinical treatment of depressive symptoms.

Affimer proteins are versatile and renewable affinity reagents

PubMed Central

Tiede, Christian; Bedford, Robert; Heseltine, Sophie J; Smith, Gina; Wijetunga, Imeshi; Ross, Rebecca; AlQallaf, Danah; Roberts, Ashley PE; Balls, Alexander; Curd, Alistair; Hughes, Ruth E; Martin, Heather; Needham, Sarah R; Zanetti-Domingues, Laura C; Sadigh, Yashar; Peacock, Thomas P; Tang, Anna A; Gibson, Naomi; Kyle, Hannah; Platt, Geoffrey W; Ingram, Nicola; Taylor, Thomas; Coletta, Louise P; Manfield, Iain; Knowles, Margaret; Bell, Sandra; Esteves, Filomena; Maqbool, Azhar; Prasad, Raj K; Drinkhill, Mark; Bon, Robin S; Patel, Vikesh; Goodchild, Sarah A; Martin-Fernandez, Marisa; Owens, Ray J; Nettleship, Joanne E; Webb, Michael E; Harrison, Michael; Lippiat, Jonathan D; Ponnambalam, Sreenivasan; Peckham, Michelle; Smith, Alastair; Ferrigno, Paul Ko; Johnson, Matt; McPherson, Michael J; Tomlinson, Darren Charles

2017-01-01

Molecular recognition reagents are key tools for understanding biological processes and are used universally by scientists to study protein expression, localisation and interactions. Antibodies remain the most widely used of such reagents and many show excellent performance, although some are poorly characterised or have stability or batch variability issues, supporting the use of alternative binding proteins as complementary reagents for many applications. Here we report on the use of Affimer proteins as research reagents. We selected 12 diverse molecular targets for Affimer selection to exemplify their use in common molecular and cellular applications including the (a) selection against various target molecules; (b) modulation of protein function in vitro and in vivo; (c) labelling of tumour antigens in mouse models; and (d) use in affinity fluorescence and super-resolution microscopy. This work shows that Affimer proteins, as is the case for other alternative binding scaffolds, represent complementary affinity reagents to antibodies for various molecular and cell biology applications. DOI: http://dx.doi.org/10.7554/eLife.24903.001 PMID:28654419

Analysis of conjugation of chloramphenicol and hemoglobin by fluorescence, circular dichroism and molecular modeling

NASA Astrophysics Data System (ADS)

Ding, Fei; Liu, Wei; Sun, Ye; Yang, Xin-Ling; Sun, Ying; Zhang, Li

2012-01-01

Chloramphenicol is a low cost, broad spectrum, highly active antibiotic, and widely used in the treatment of serious infections, including typhoid fever and other life-threatening infections of the central nervous system and respiratory tract. The purpose of the present study was to examine the conjugation of chloramphenicol with hemoglobin (Hb) and compared with albumin at molecular level, utilizing fluorescence, UV/vis absorption, circular dichroism (CD) as well as molecular modeling. Fluorescence data indicate that drug bind Hb generate quenching via static mechanism, this corroborates UV/vis absorption measurements that the ground state complex formation with an affinity of 10 4 M -1, and the driving forces in the Hb-drug complex are hydrophilic interactions and hydrogen bonds, as derived from computational model. The accurate binding site of drug has been identified from the analysis of fluorescence and molecular modeling, α1β2 interface of Hb was assigned to possess high-affinity for drug, which located at the β-37 Trp nearby. The structural investigation of the complexed Hb by synchronous fluorescence, UV/vis absorption, and CD observations revealed some degree of Hb structure unfolding upon complexation. Based on molecular modeling, we can draw the conclusion that the binding affinity of drug with albumin is superior, compared with Hb. These phenomena can provide salient information on the absorption, distribution, pharmacology, and toxicity of chloramphenicol and other drugs which have analogous configuration with chloramphenicol.

Quantum-chemical, NMR, FT IR, and ESI MS studies of complexes of colchicine with Zn(II).

PubMed

Jankowski, Wojciech; Kurek, Joanna; Barczyński, Piotr; Hoffmann, Marcin

2017-04-01

Colchicine is a tropolone alkaloid from Colchicinum autumnale. It shows antifibrotic, antimitotic, and anti-inflammatory activities, and is used to treat gout and Mediterranean fever. In this work, complexes of colchicine with zinc(II) nitrate were synthesized and investigated using DFT, 1 H and 13 C NMR, FT IR, and ESI MS. The counterpoise-corrected and uncorrected interaction energies of these complexes were calculated. We also calculated their 1 H, 13 C NMR, and IR spectra and compared them with the corresponding experimentally obtained data. According to the ESI MS mass spectra, colchicine forms stable complexes with zinc(II) nitrate that have various stoichiometries: 2:1, 1:1:1, and 2:1:1 with respect to colchichine, Zn(II), and nitrate ion. All of the complexes were investigated using the quantum theory of atoms in molecules (QTAIM). The calculated and the measured spectra showed differences before and after the complexation process. Calculated electron densities and bond critical points indicated the presence of bonds between the ligands and the central cation in the investigated complexes that satisfied the quantum theory of atoms in molecules. Graphical Abstract DFT, NMR, FT IR, ESI MS, QTAIM and puckering studies of complexes of colchicine with Zn(II).

Highly selective removal of Zn(II) ion from hot-dip galvanizing pickling waste with amino-functionalized Fe3O4@SiO2 magnetic nano-adsorbent.

PubMed

Bao, Shuangyou; Tang, Lihong; Li, Kai; Ning, Ping; Peng, Jinhui; Guo, Huibin; Zhu, Tingting; Liu, Ye

2016-01-15

Amino-functionalized Fe3O4@SiO2 magnetic nano-adsorbent was used as a novel sorbent to highly selective removal of Zn(II) ion from hot-dip galvanizing pickling waste in the presence of Fe(II). These hot-dip galvanizing pickling waste mainly contain ZnCl2 and FeCl2 in aqueous HCl media. The properties of this magnetic adsorbent were examined by transmission electron microscopy (TEM), powder X-ray diffraction (XRD), infrared spectrometer (FT-IR) and BET surface area measurements. Various factors influencing the adsorption of Zn(II) ion such as initial concentration of metal ions, the amount of adsorbent, pH value of the solutions, the concentration of coexisting iron ion were investigated by batch experiments. The results indicated that the adsorption equilibrium data obeyed the Freundlich model with maximum adsorption capacities for Zn(II) to 169.5mg/g. The maximum adsorption occurred at pH 5±0.1 and Fe(II) interferences had no obvious influence. This work provides a potential and unique technique for zinc ion removal from hot-dip galvanizing pickling waste. Copyright © 2015 Elsevier Inc. All rights reserved.

Fluorescence Imaging/Agents in Tumor Resection.

PubMed

Stummer, Walter; Suero Molina, Eric

2017-10-01

Intraoperative fluorescence imaging allows real-time identification of diseased tissue during surgery without being influenced by brain shift and surgery interruption. 5-Aminolevulinic acid, useful for malignant gliomas and other tumors, is the most broadly explored compound approved for fluorescence-guided resection. Intravenous fluorescein sodium has recently received attention, highlighting tumor tissue based on extravasation at the blood-brain barrier (defective in many brain tumors). Fluorescein in perfused brain, unselective extravasation in brain perturbed by surgery, and propagation with edema are concerns. Fluorescein is not approved but targeted fluorochromes with affinity to brain tumor cells, in development, may offer future advantages. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Comparative Study of the Fatty Acid Binding Process of a New FABP from Cherax quadricarinatus by Fluorescence Intensity, Lifetime and Anisotropy

PubMed Central

Li, Jiayao; Henry, Etienne; Wang, Lanmei; Delelis, Olivier; Wang, Huan; Simon, Françoise; Tauc, Patrick; Brochon, Jean-Claude; Zhao, Yunlong; Deprez, Eric

2012-01-01

Fatty acid-binding proteins (FABPs) are small cytosolic proteins, largely distributed in invertebrates and vertebrates, which accomplish uptake and intracellular transport of hydrophobic ligands such as fatty acids. Although long chain fatty acids play multiple crucial roles in cellular functions (structural, energy metabolism, regulation of gene expression), the precise functions of FABPs, especially those of invertebrate species, remain elusive. Here, we have identified and characterized a novel FABP family member, Cq-FABP, from the hepatopancreas of red claw crayfish Cherax quadricarinatus. We report the characterization of fatty acid-binding affinity of Cq-FABP by four different competitive fluorescence-based assays. In the two first approaches, the fluorescent probe 8-Anilino-1-naphthalenesulfonate (ANS), a binder of internal cavities of protein, was used either by directly monitoring its fluorescence emission or by monitoring the fluorescence resonance energy transfer occurring between the single tryptophan residue of Cq-FABP and ANS. The third and the fourth approaches were based on the measurement of the fluorescence emission intensity of the naturally fluorescent cis-parinaric acid probe or the steady-state fluorescence anisotropy measurements of a fluorescently labeled fatty acid (BODIPY-C16), respectively. The four methodologies displayed consistent equilibrium constants for a given fatty acid but were not equivalent in terms of analysis. Indeed, the two first methods were complicated by the existence of non specific binding modes of ANS while BODIPY-C16 and cis-parinaric acid specifically targeted the fatty acid binding site. We found a relationship between the affinity and the length of the carbon chain, with the highest affinity obtained for the shortest fatty acid, suggesting that steric effects primarily influence the interaction of fatty acids in the binding cavity of Cq-FABP. Moreover, our results show that the binding affinities of several fatty

Glycolipid acquisition by human glycolipid transfer protein dramatically alters intrinsic tryptophan fluorescence: insights into glycolipid binding affinity.

PubMed

Zhai, Xiuhong; Malakhova, Margarita L; Pike, Helen M; Benson, Linda M; Bergen, H Robert; Sugár, István P; Malinina, Lucy; Patel, Dinshaw J; Brown, Rhoderick E

2009-05-15

Glycolipid transfer proteins (GLTPs) are small, soluble proteins that selectively accelerate the intermembrane transfer of glycolipids. The GLTP fold is conformationally unique among lipid binding/transfer proteins and serves as the prototype and founding member of the new GLTP superfamily. In the present study, changes in human GLTP tryptophan fluorescence, induced by membrane vesicles containing glycolipid, are shown to reflect glycolipid binding when vesicle concentrations are low. Characterization of the glycolipid-induced "signature response," i.e. approximately 40% decrease in Trp intensity and approximately 12-nm blue shift in emission wavelength maximum, involved various modes of glycolipid presentation, i.e. microinjection/dilution of lipid-ethanol solutions or phosphatidylcholine vesicles, prepared by sonication or extrusion and containing embedded glycolipids. High resolution x-ray structures of apo- and holo-GLTP indicate that major conformational alterations are not responsible for the glycolipid-induced GLTP signature response. Instead, glycolipid binding alters the local environment of Trp-96, which accounts for approximately 70% of total emission intensity of three Trp residues in GLTP and provides a stacking platform that aids formation of a hydrogen bond network with the ceramide-linked sugar of the glycolipid headgroup. The changes in Trp signal were used to quantitatively assess human GLTP binding affinity for various lipids including glycolipids containing different sugar headgroups and homogenous acyl chains. The presence of the glycolipid acyl chain and at least one sugar were essential for achieving a low-to-submicromolar dissociation constant that was only slightly altered by increased sugar headgroup complexity.

Interactions of Zn(II) Ions with Humic Acids Isolated from Various Type of Soils. Effect of pH, Zn Concentrations and Humic Acids Chemical Properties.

PubMed

Boguta, Patrycja; Sokołowska, Zofia

2016-01-01

The main aim of this study was the analysis of the interaction between humic acids (HAs) from different soils and Zn(II) ions at wide concentration ranges and at two different pHs, 5 and 7, by using fluorescence and FTIR spectroscopy, as well as potentiometric measurements. The presence of a few areas of HAs structures responsible for Zn(II) complexing was revealed. Complexation at α-sites (low humified structures of low-molecular weight and aromatic polycondensation) and β-sites (weakly humified structures) was stronger at pH 7 than 5. This trend was not observed for γ-sites (structures with linearly-condensed aromatic rings, unsaturated bonds and large molecular weight). The amount of metal complexed at pH5 and 7 by α and γ-structures increased with a decrease in humification and aromaticity of HAs, contrary to β-areas where complexation increased with increasing content of carboxylic groups. The stability of complexes was higher at pH 7 and was the highest for γ-structures. At pH 5, stability decreased with C/N increase for α-areas and -COOH content increase for β-sites; stability increased with humification decrease for γ-structures. The stability of complexes at α and β-areas at pH 7 decreased with a drop in HAs humification. FTIR spectra at pH 5 revealed that the most-humified HAs tended to cause bidentate bridging coordination, while in the case of the least-humified HAs, Zn caused bidentate bridging coordination at low Zn additions and bidentate chelation at the highest Zn concentrations. Low Zn doses at pH 7 caused formation of unidentate complexes while higher Zn doses caused bidentate bridging. Such processes were noticed for HAs characterized by high oxidation degree and high oxygen functional group content; where these were low, HAs displayed bidentate bridging or even bidentate chelation. To summarize, the above studies have showed significant impact of Zn concentration, pH and some properties of HAs on complexation reactions of humic

Interactions of Zn(II) Ions with Humic Acids Isolated from Various Type of Soils. Effect of pH, Zn Concentrations and Humic Acids Chemical Properties

PubMed Central

Boguta, Patrycja; Sokołowska, Zofia

2016-01-01

The main aim of this study was the analysis of the interaction between humic acids (HAs) from different soils and Zn(II) ions at wide concentration ranges and at two different pHs, 5 and 7, by using fluorescence and FTIR spectroscopy, as well as potentiometric measurements. The presence of a few areas of HAs structures responsible for Zn(II) complexing was revealed. Complexation at α-sites (low humified structures of low-molecular weight and aromatic polycondensation) and β-sites (weakly humified structures) was stronger at pH 7 than 5. This trend was not observed for γ-sites (structures with linearly-condensed aromatic rings, unsaturated bonds and large molecular weight). The amount of metal complexed at pH5 and 7 by α and γ-structures increased with a decrease in humification and aromaticity of HAs, contrary to β-areas where complexation increased with increasing content of carboxylic groups. The stability of complexes was higher at pH 7 and was the highest for γ-structures. At pH 5, stability decreased with C/N increase for α-areas and -COOH content increase for β-sites; stability increased with humification decrease for γ-structures. The stability of complexes at α and β-areas at pH 7 decreased with a drop in HAs humification. FTIR spectra at pH 5 revealed that the most-humified HAs tended to cause bidentate bridging coordination, while in the case of the least-humified HAs, Zn caused bidentate bridging coordination at low Zn additions and bidentate chelation at the highest Zn concentrations. Low Zn doses at pH 7 caused formation of unidentate complexes while higher Zn doses caused bidentate bridging. Such processes were noticed for HAs characterized by high oxidation degree and high oxygen functional group content; where these were low, HAs displayed bidentate bridging or even bidentate chelation. To summarize, the above studies have showed significant impact of Zn concentration, pH and some properties of HAs on complexation reactions of humic

Fluorescence resonance energy transfer between green fluorescent protein and doxorubicin enabled by DNA nanotechnology.

PubMed

Heger, Zbynek; Kominkova, Marketa; Cernei, Natalia; Krejcova, Ludmila; Kopel, Pavel; Zitka, Ondrej; Adam, Vojtech; Kizek, Rene

2014-12-01

DNA nanotechnology is a rapidly growing research area, where DNA may be used for wide range of applications such as construction of nanodevices serving for large scale of diverse purposes. Likewise a panel of various purified fluorescent proteins is investigated for their ability to emit their typical fluorescence spectra under influence of particular excitation. Hence these proteins may form ideal donor molecules for assembly of fluorescence resonance emission transfer (FRET) constructions. To extend the application possibilities of fluorescent proteins, while using DNA nanotechnology, we developed nanoconstruction comprising green fluorescent protein (GFP) bound onto surface of surface active nanomaghemite and functionalized with gold nanoparticles. We took advantage of natural affinity between gold and thiol moieties, which were modified to bind DNA fragment. Finally we enclosed doxorubicin into fullerene cages. Doxorubicin intercalated in DNA fragment bound on the particles and thus we were able to connect these parts together. Because GFP behaved as a donor and doxorubicin as an acceptor using excitation wavelength for GFP (395 nm) in emission wavelength of doxorubicin (590 nm) FRET was observed. This nanoconstruction may serve as a double-labeled transporter of doxorubicin guided by force of external magnetic force owing to the presence of nanomaghemite. Further nanomaghemite offers the possibility of using this technology for thermotherapy. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Genome-wide analysis of the Zn(II)2Cys6 zinc cluster-encoding gene family in Aspergillus flavus

USDA-ARS?s Scientific Manuscript database

Proteins with a Zn(II)2Cys6 domain, Cys-X2-Cys-X6-Cys-X5-12-Cys-X2-Cys-X6-9-Cys (hereafter, referred to as the C6 domain), form a subclass of zinc finger proteins found exclusively in fungi and yeast. Genome sequence databases of Saccharomyces cerevisiae and Candida albicans have provided an overvie...

A Zn(II)2Cys6 DNA binding protein regulates the sirodesmin PL biosynthetic gene cluster in Leptosphaeria maculans

PubMed Central

Fox, Ellen M.; Gardiner, Donald M.; Keller, Nancy P.; Howlett, Barbara J.

2008-01-01

A gene, sirZ, encoding a Zn(II)2Cys6 DNA binding protein is present in a cluster of genes responsible for the biosynthesis of the epipolythiodioxopiperazine (ETP) toxin, sirodesmin PL in the ascomycete plant pathogen, Leptosphaeria maculans. RNA-mediated silencing of sirZ gives rise to transformants that produce only residual amounts of sirodesmin PL and display a decrease in the transcription of several sirodesmin PL biosynthetic genes. This indicates that SirZ is a major regulator of this gene cluster. Proteins similar to SirZ are encoded in the gliotoxin biosynthetic gene cluster of Aspergillus fumigatus (gliZ) and in an ETP-like cluster in Penicillium lilacinoechinulatum (PlgliZ). Despite its high level of sequence similarity to gliZ, PlgliZ is unable to complement the gliotoxin-deficiency of a mutant of gliZ in A. fumigatus. Putative binding sites for these regulatory proteins in the promoters of genes in these clusters were predicted using bioinformatic analysis. These sites are similar to those commonly bound by other proteins with Zn(II)2Cys6 DNA binding domains. PMID:18023597

Hypothesis testing for the validation of the kinetic spectrophotometric methods for the determination of lansoprazole in bulk and drug formulations via Fe(III) and Zn(II) chelates.

PubMed

Rahman, Nafisur; Kashif, Mohammad

2010-03-01

Point and interval hypothesis tests performed to validate two simple and economical, kinetic spectrophotometric methods for the assay of lansoprazole are described. The methods are based on the formation of chelate complex of the drug with Fe(III) and Zn(II). The reaction is followed spectrophotometrically by measuring the rate of change of absorbance of coloured chelates of the drug with Fe(III) and Zn(II) at 445 and 510 nm, respectively. The stoichiometric ratio of lansoprazole to Fe(III) and Zn(II) complexes were found to be 1:1 and 2:1, respectively. The initial-rate and fixed-time methods are adopted for determination of drug concentrations. The calibration graphs are linear in the range 50-200 µg ml⁻¹ (initial-rate method), 20-180 µg ml⁻¹ (fixed-time method) for lansoprazole-Fe(III) complex and 120-300 (initial-rate method), and 90-210 µg ml⁻¹ (fixed-time method) for lansoprazole-Zn(II) complex. The inter-day and intra-day precision data showed good accuracy and precision of the proposed procedure for analysis of lansoprazole. The point and interval hypothesis tests indicate that the proposed procedures are not biased. Copyright © 2010 John Wiley & Sons, Ltd.

Fluorescence Quenching Property of C-Phycocyanin from Spirulina platensis and its Binding Efficacy with Viable Cell Components.

PubMed

Paswan, Meenakshi B; Chudasama, Meghna M; Mitra, Madhusree; Bhayani, Khushbu; George, Basil; Chatterjee, Shruti; Mishra, Sandhya

2016-03-01

Phycocyanin is a natural brilliant blue colored, fluorescent protein, which is commonly present in cyanobacteria. In this study, C-phycocyanin was extracted and purified from Spirulina platensis, which are multicellular and filamentous cyanobacteria of greater importance because of its various biological and pharmacological potential. It was analyzed for its binding affinity towards blood cells, algal cells, genomic DNA of microalgae, and bacteria at different temperature and incubation time. It showed good binding affinity with these components even at low concentration of 2.5 μM. The purpose of this study was to evaluate the applicability of C-phycocyanin as a green fluorescent dye substituting carcinogenic chemical dyes.

Fluorescence Lifetime Imaging of Physiological Free Cu(II) Levels in Live Cells with a Cu(II)-Selective Carbonic Anhydrase-Based Biosensor

PubMed Central

McCranor, Bryan J.; Szmacinski, Henryk; Zeng, Hui Hui; Stoddard, A.K.; Hurst, Tamiika; Fierke, Carol A.; Lakowicz, J.R.

2014-01-01

Copper is a required trace element that plays key roles in a number of human enzymes, such that copper deficiency or genetic defects in copper transport lead to serious or fatal disease. Rae, et al., had famously predicted that free copper ion levels in the cell cytoplasm were extremely low, typically too low to be observable. We recently developed a variant of human apocarbonic anhydrase II for sensing metal ions that exhibits 25-fold better selectivity for Cu(II) over Zn(II) than the wild type protein, enabling us to accurately measure Cu(II) in the presence of ordinary cellular (picomolar) concentrations of free zinc. We inserted a fluorescent labeled Cu(II)-specific variant of human apocarbonic anhydrase into PC-12 cells and found that the levels are indeed extremely low (in the femtomolar range). We imaged the free Cu(II) levels in living cells by means of frequency-domain fluorescence lifetime microscopy. Implications of this finding are discussed. PMID:24671220

Proflavine acts as a Rev inhibitor by targeting the high-affinity Rev binding site of the Rev responsive element of HIV-1.

PubMed

DeJong, Eric S; Chang, Chia-en; Gilson, Michael K; Marino, John P

2003-07-08

Rev is an essential regulatory HIV-1 protein that binds the Rev responsive element (RRE) within the env gene of the HIV-1 RNA genome, activating the switch between viral latency and active viral replication. Previously, we have shown that selective incorporation of the fluorescent probe 2-aminopurine (2-AP) into a truncated form of the RRE sequence (RRE-IIB) allowed the binding of an arginine-rich peptide derived from Rev and aminoglycosides to be characterized directly by fluorescence methods. Using these fluorescence and nuclear magnetic resonance (NMR) methods, proflavine has been identified, through a limited screen of selected small heterocyclic compounds, as a specific and high-affinity RRE-IIB binder which inhibits the interaction of the Rev peptide with RRE-IIB. Direct and competitive 2-AP fluorescence binding assays reveal that there are at least two classes of proflavine binding sites on RRE-IIB: a high-affinity site that competes with the Rev peptide for binding to RRE-IIB (K(D) approximately 0.1 +/- 0.05 microM) and a weaker binding site(s) (K(D) approximately 1.1 +/- 0.05 microM). Titrations of RRE-IIB with proflavine, monitored using (1)H NMR, demonstrate that the high-affinity proflavine binding interaction occurs with a 2:1 (proflavine:RRE-IIB) stoichiometry, and NOEs observed in the NOESY spectrum of the 2:1 proflavine.RRE-IIB complex indicate that the two proflavine molecules bind specifically and close to each other within a single binding site. NOESY data further indicate that formation of the 2:1 proflavine.RRE-IIB complex stabilizes base pairing and stacking within the internal purine-rich bulge of RRE-IIB in a manner analogous to what has been observed in the Rev peptide.RRE-IIB complex. The observation that proflavine competes with Rev for binding to RRE-IIB by binding as a dimer to a single high-affinity site opens the possibility for rational drug design based on linking and modifying it and related compounds.

Binding patterns and structure-affinity relationships of food azo dyes with lysozyme: a multitechnique approach.

PubMed

Peng, Wei; Ding, Fei; Peng, Yu-Kui; Jiang, Yu-Ting; Zhang, Li

2013-12-18

Food dyes serve to beguile consumers: they are often used to imitate the presence of healthful, colorful food produce such as fruits and vegetables. But considering the hurtful impact of these chemicals on the human body, it is time to thoroughly uncover the toxicity of these food dyes at the molecular level. In the present contribution, we have examined the molecular reactions of protein lysozyme with model food azo compound Color Index (C.I.) Acid Red 2 and its analogues C.I. Acid Orange 52, Solvent Yellow 2, and the core structure of azobenzene using a combination of biophysical methods at physiological conditions. Fluorescence, circular dichroism (CD), time-resolved fluorescence, UV-vis absorption as well as computer-aided molecular modeling were used to analyze food dye affinity, binding mode, energy transfer, and the effects of food dye complexation on lysozyme stability and conformation. Fluorescence emission spectra indicate complex formation at 10(-5) M dye concentration, and this corroborates time-resolved fluorescence results showing the diminution in the tryptophan (Trp) fluorescence mainly via a static type (KSV = 1.505 × 10(4) M(-1)) and Förster energy transfer. Structural analysis displayed the participation of several amino acid residues in food dye protein adducts, with hydrogen bonds, π-π and cation-π interactions, but the conformation of lysozyme was unchanged in the process, as derived from fluorescence emission, far-UV CD, and synchronous fluorescence spectra. The overall affinity of food dye is 10(4) M(-1) and there exists only one kind of binding domain in protein for food dye. These data are consistent with hydrophobic probe 8-anilino-1-naphthalenesulfonic acid (ANS) displacement, and molecular modeling manifesting the food dye binding patch was near to Trp-62 and Trp-63 residues of lysozyme. On the basis of the computational analyses, we determine that the type of substituent on the azobenzene structure has a powerful influence on the

Computer-aided design of peptide near infrared fluorescent probe for tumor diagnosis

NASA Astrophysics Data System (ADS)

Zhang, Congying; Gu, Yueqing

2014-09-01

Integrin αvβ3 receptors are expressed on activated endothelial cells during neovascularization to maintain tumor growth, so they become hot research tagets in cancer diagnosis. Peptides possess several attractive features when compared to protein and small molecule, such as small size and high structural compatibility with target proteins. Efficient design of high-affinity peptide ligands to Integrin αvβ3 receptors has been an important problem. Designed peptides in silico provide a valuable and high-selectivity peptide, meanwhile decrease the time of drug screening. In this study, we design peptide which can bind with integrin αvβ3 via computer, and then synthesis near infrared fluorescent probe. The characterization of this near infrared fluorescent probe was detected by UV. To investigate the tumor cell targeting of this probe, it was labeled with visible fluorescent dye Rhodamine B (RhB) for microscopy. To evaluate the targeting capability of this near infrared fluorescent probe, mice bearing integrin αvβ3 positive tumor xenografts were used. In vitro cellular experiments indicated that this probe have a clear binding affinity to αvβ3-positive tumor cells. In vivo experiments confirmed the receptor binding specificity of this probe. The peptide of computational design can bind with integrin αvβ3. Combined peptide near-infrared fluorescent probe with imaging technology use for clinical and tumor diagnosis have a greater development in future.

High affinity receptor labeling based on basic leucine zipper domain peptides conjugated with pH-sensitive fluorescent dye: Visualization of AMPA-type glutamate receptor endocytosis in living neurons.

PubMed

Hayashi, Ayako; Asanuma, Daisuke; Kamiya, Mako; Urano, Yasuteru; Okabe, Shigeo

2016-01-01

Techniques to visualize receptor trafficking in living neurons are important, but currently available methods are limited in their labeling efficiency, specificity and reliability. Here we report a method for receptor labeling with a basic leucine zipper domain peptide (ZIP) and a binding cassette specific to ZIP. Receptors are tagged with a ZIP-binding cassette at their extracellular domain. Tagged receptors expressed in cultured cells were labeled with exogenously applied fluorescently labeled ZIP with low background and high affinity. To test if ZIP labeling is useful in monitoring endocytosis and intracellular trafficking, we next conjugated ZIP with a pH-sensitive dye RhP-M (ZIP-RhP-M). ZIP binding to its binding cassette was pH-resistant and RhP-M fluorescence dramatically increased in acidic environment. Thus AMPA-type glutamate receptors (AMPARs) labeled by ZIP-RhP-M can report receptor endocytosis and subsequent intracellular trafficking. Application of ZIP-RhP-M to cultured hippocampal neurons expressing AMPARs tagged with a ZIP-binding cassette resulted in appearance of fluorescent puncta in PSD-95-positive large spines, suggesting local endocytosis and acidification of AMPARs in individual mature spines. This spine pool of AMPARs in acidic environment was distinct from the early endosomes labeled by transferrin uptake. These results suggest that receptor labeling by ZIP-RhP-M is a useful technique for monitoring endocytosis and intracellular trafficking. This article is part of the Special Issue entitled 'Synaptopathy--from Biology to Therapy'. Copyright © 2015 Elsevier Ltd. All rights reserved.

Breaking the color barrier - a multi-selective antibody reporter offers innovative strategies of fluorescence detection.

PubMed

Gallo, Eugenio; Jarvik, Jonathan W

2017-08-01

A novel bi-partite fluorescence platform exploits the high affinity and selectivity of antibody scaffolds to capture and activate small-molecule fluorogens. In this report, we investigated the property of multi-selectivity activation by a single antibody against diverse cyanine family fluorogens. Our fluorescence screen identified three cell-impermeant fluorogens, each with unique emission spectra (blue, green and red) and nanomolar affinities. Most importantly, as a protein fusion tag to G-protein-coupled receptors, the antibody biosensor retained full activity - displaying bright fluorogen signals with minimal background on live cells. Because fluorogen-activating antibodies interact with their target ligands via non-covalent interactions, we were able to perform advanced multi-color detection strategies on live cells, previously difficult or impossible with conventional reporters. We found that by fine-tuning the concentrations of the different color fluorogen molecules in solution, a user may interchange the fluorescence signal (onset versus offset), execute real-time signal exchange via fluorogen competition, measure multi-channel fluorescence via co-labeling, and assess real-time cell surface receptor traffic via pulse-chase experiments. Thus, here we inform of an innovative reporter technology based on tri-color signal that allows user-defined fluorescence tuning in live-cell applications. © 2017. Published by The Company of Biologists Ltd.

Fluorescent 2D WS2 Nanosheets Bearing Chemical Affinity Elements for the Recognition of Glycated Hemoglobin.

PubMed

Yang, Jin-Kyoung; Lee, Hye-Rim; Hwang, In-Jun; Kim, Hye-In; Yim, DaBin; Kim, Jong-Ho

2018-05-14

It is required to exfoliate and functionalize 2D transition metal dichalcogenides (TMDs) in an aqueous solution for biological and medical applications. Herein, the approach for the simultaneous exfoliation and functionalization of 2D WS 2 nanosheets using boronic acid-modified poly(vinyl alcohol) (B-PVA) in an aqueous solution is reported, and the B-PVA-functionalized WS 2 nanosheets (B-PVA-WS 2 ) are exploited as a fluorescent biosensor for the detection of glycated hemoglobin, HbA1c. The synthetic B-PVA polymer facilitates the exfoliation and functionalization of WS 2 nanosheets from the bulk counterpart in the aqueous solution via a pulsed sonication process, resulting in fluorescent B-PVA-WS 2 nanohybrids with a specific recognition of HbA1c. The fluorescence of the B-PVA-WS 2 is quenched in the presence of HbA1c, whereas PVA-functionalized WS 2 (PVA-WS 2 ), not bearing boronic acid as a recognition moiety, shows no fluorescence changes upon the addition of the target. The B-PVA-WS 2 is able to selectively detect HbA1c at the concentration as low as 3.3 × 10 -8 m based on its specific fluorescence quenching. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Thirty-fifth anniversary of the optical affinity sensor for glucose: a personal retrospective.

PubMed

Schultz, Jerome S

2015-01-01

Since 1962 when Clark introduced the enzyme electrode, research has been intense for a robust implantable glucose sensor. An alternative "optical affinity sensor" was introduced by Jerome Schultz in 1979. The evolution of this sensor technology into a new methodology is reviewed. The approach integrates a variety of disparate concepts: the selectivity of immunoassays-selectivity for glucose was obtained with concanavalin A, detection sensitivity was obtained with fluorescence (FITC-Dextran), and miniaturization was achieved by the use of an optical fiber readout system. Refinements of Schultz's optical affinity sensor approach over the past 35 years have led to a number of configurations that show great promise to meet the needs of a successful implantable continuous monitoring device for diabetics, some of which are currently being tested clinically. © 2014 Diabetes Technology Society.

Two-dimensional Zn(II) and one-dimensional Co(II) coordination polymers based on benzene-1,4-dicarboxylate and pyridine ligands.

PubMed

Zhou, Li-Juan; Han, Chang-Bao; Wang, Yu-Ling

2016-02-01

Coordination polymers constructed from metal ions and organic ligands have attracted considerable attention owing to their diverse structural topologies and potential applications. Ligands containing carboxylate groups are among the most extensively studied because of their versatile coordination modes. Reactions of benzene-1,4-dicarboxylic acid (H2BDC) and pyridine (py) with Zn(II) or Co(II) yielded two new coordination polymers, namely, poly[(μ4-benzene-1,4-dicarboxylato-κ(4)O:O':O'':O''')(pyridine-κN)zinc(II)], [Zn(C8H4O2)(C5H5N)]n, (I), and catena-poly[aqua(μ3-benzene-1,4-dicarboxylato-κ(3)O:O':O'')bis(pyridine-κN)cobalt(II)], [Co(C8H4O2)(C5H5N)2(H2O)]n, (II). In compound (I), the Zn(II) cation is five-coordinated by four carboxylate O atoms from four BDC(2-) ligands and one pyridine N atom in a distorted square-pyramidal coordination geometry. Four carboxylate groups bridge two Zn(II) ions to form centrosymmetric paddle-wheel-like Zn2(μ2-COO)4 units, which are linked by the benzene rings of the BDC(2-) ligands to generate a two-dimensional layered structure. The two-dimensional layer is extended into a three-dimensional supramolecular structure with the help of π-π stacking interactions between the aromatic rings. Compound (II) has a one-dimensional double-chain structure based on Co2(μ2-COO)2 units. The Co(II) cations are bridged by BDC(2-) ligands and are octahedrally coordinated by three carboxylate O atoms from three BDC(2-) ligands, one water O atom and two pyridine N atoms. Interchain O-H...O hydrogen-bonding interactions link these chains to form a three-dimensional supramolecular architecture.

Lp-dual affine surface area

NASA Astrophysics Data System (ADS)

Wei, Wang; Binwu, He

2008-12-01

According to the notion of Lp-affine surface area by Lutwak, in this paper, we introduce the concept of Lp-dual affine surface area. Further, we establish the affine isoperimetric inequality and the Blaschke-Santaló inequality for Lp-dual affine surface area. Besides, the dual Brunn-Minkowski inequality for Lp-dual affine surface area is presented.

Use of a benzimidazole derivative BF-188 in fluorescence multispectral imaging for selective visualization of tau protein fibrils in the Alzheimer's disease brain.

PubMed

Harada, Ryuichi; Okamura, Nobuyuki; Furumoto, Shozo; Yoshikawa, Takeo; Arai, Hiroyuki; Yanai, Kazuhiko; Kudo, Yukitsuka

2014-02-01

Selective visualization of amyloid-β and tau protein deposits will help to understand the pathophysiology of Alzheimer's disease (AD). Here, we introduce a novel fluorescent probe that can distinguish between these two deposits by multispectral fluorescence imaging technique. Fluorescence spectral analysis was performed using AD brain sections stained with novel fluorescence compounds. Competitive binding assay using [(3)H]-PiB was performed to evaluate the binding affinity of BF-188 for synthetic amyloid-β (Aβ) and tau fibrils. In AD brain sections, BF-188 clearly stained Aβ and tau protein deposits with different fluorescence spectra. In vitro binding assays indicated that BF-188 bound to both amyloid-β and tau fibrils with high affinity (K i  < 10 nM). In addition, BF-188 showed an excellent blood-brain barrier permeability in mice. Multispectral imaging with BF-188 could potentially be used for selective in vivo imaging of tau deposits as well as amyloid-β in the brain.

Complementary DNA display selection of high-affinity peptides binding the vacuolating toxin (VacA) of Helicobacter pylori.

PubMed

Hayakawa, Yumiko; Matsuno, Mitsuhiro; Tanaka, Makoto; Wada, Akihiro; Kitamura, Koichiro; Takei, Osamu; Sasaki, Ryuzo; Mizukami, Tamio; Hasegawa, Makoto

2015-09-01

Artificial peptides designed for molecular recognition of a bacterial toxin have been developed. Vacuolating cytotoxin A protein (VacA) is a major virulence factor of Helicobacter pylori, a gram-negative microaerophilic bacterium inhabiting the upper gastrointestinal tract, particularly the stomach. This study attempted to identify specific peptide sequences with high affinity for VacA using systematic directed evolution in vitro, a cDNA display method. A surface plasmon resonance-based biosensor and fluorescence correlation spectroscopy to examine binding of peptides with VacA identified a peptide (GRVNQRL) with high affinity. Cyclization of the peptide by attaching cysteine residues to both termini improved its binding affinity to VacA, with a dissociation constant (Kd ) of 58 nm. This study describes a new strategy for the development of artificial functional peptides, which are promising materials in biochemical analyses and medical applications. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

Purification of yeast alcohol dehydrogenase by using immobilized metal affinity cryogels.

PubMed

Akduman, Begüm; Uygun, Murat; Uygun, Deniz Aktaş; Akgöl, Sinan; Denizli, Adil

2013-12-01

In this study, poly(2-hydroxyethyl methacrylate-glycidylmethacrylate) [poly(HEMA-GMA)] cryogels were prepared by radical cryocopolymerization of HEMA with GMA as a functional comonomer and N,N'-methylene-bisacrylamide (MBAAm) as a crosslinker. Iminodiacetic acid (IDA) functional groups were attached via ring opening of the epoxy group on the poly(HEMA-GMA) cryogels and then Zn(II) ions were chelated with these structures. Characterization of cryogels was performed by FTIR, SEM, EDX and swelling studies. These cryogels have interconnected pores of 30-50 μm size. The equilibrium swelling degree of Zn(II) chelated poly(HEMA-GMA)-IDA cryogels was approximately 600%. Zn(II) chelated poly(HEMA-GMA)-IDA cryogels were used in the adsorption of alcohol dehydrogenase from aqueous solutions and adsorption was performed in continuous system. The effects of pH, alcohol dehydrogenase concentration, temperature, and flow rate on adsorption were investigated. The maximum amount of alcohol dehydrogenase adsorption was determined to be 9.94 mg/g cryogel at 1.0mg/mL alcohol dehydrogenase concentration and in acetate buffer at pH5.0 with a flow rate of 0.5 mL/min. Desorption of adsorbed alcohol dehydrogenase was carried out by using 1.0M NaCI at pH8.0 phosphate buffer and desorption yield was found to be 93.5%. Additionally, these cryogels were used for purification of alcohol dehydrogenase from yeast with a single-step. The purity of desorbed alcohol dehydrogenase was shown by silver-stained SDS-PAGE. This purification process can successfully be used for the purification of alcohol dehydrogenase from unclarified yeast homogenates and this work is the first report about the usage of the cryogels for purification of alcohol dehydrogenase. © 2013 Elsevier B.V. All rights reserved.

Quantitative Fluorescence Studies in Living Cells: Extending Fluorescence Fluctuation Spectroscopy to Peripheral Membrane Proteins

NASA Astrophysics Data System (ADS)

Smith, Elizabeth Myhra

The interactions of peripheral membrane proteins with both membrane lipids and proteins are vital for many cellular processes including membrane trafficking, cellular signaling, and cell growth/regulation. Building accurate biophysical models of these processes requires quantitative characterization of the behavior of peripheral membrane proteins, yet methods to quantify their interactions inside living cells are very limited. Because peripheral membrane proteins usually exist both in membrane-bound and cytoplasmic forms, the separation of these two populations is a key challenge. This thesis aims at addressing this challenge by extending fluorescence fluctuation spectroscopy (FFS) to simultaneously measure the oligomeric state of peripheral membrane proteins in the cytoplasm and at the plasma membrane. We developed a new method based on z-scan FFS that accounts for the fluorescence contributions from cytoplasmic and membrane layers by incorporating a fluorescence intensity z-scan through the cell. H-Ras-EGFP served as a model system to demonstrate the feasibility of the technique. The resolvability and stability of z-scanning was determined as well as the oligomeric state of H-Ras-EGFP at the plasma membrane and in the cytoplasm. Further, we successfully characterized the binding affinity of a variety of proteins to the plasma membrane by quantitative analysis of the z-scan fluorescence intensity profile. This analysis method, which we refer to as z-scan fluorescence profile deconvoution, was further used in combination with dual-color competition studies to determine the lipid specificity of protein binding. Finally, we applied z-scan FFS to provide insight into the early assembly steps of the HTLV-1 retrovirus.

Human serum albumin binding assay based on displacement of a non selective fluorescent inhibitor.

PubMed

Thorarensen, Atli; Sarver, Ronald W; Tian, Fang; Ho, Andrea; Romero, Donna L; Marotti, Keith R

2007-08-15

In this paper, we describe a fluorescent antibacterial analog, 6, with utility as a competition probe to determine affinities of other antibacterial analogs for human serum albumin (HSA). Analog 6 bound to HSA with an affinity of 400+/-100 nM and the fluorescence was environmentally sensitive. With 370 nm excitation, environmental sensitivity was indicated by a quenching of the 530 nm emission when the probe bound to HSA. Displacement of dansylsarcosine from HSA by 6 indicated it competed with compounds that bound at site II (ibuprofen binding site) on HSA. Analog 6 also shifted the NMR peaks of an HSA bound oleic acid molecule that itself was affected by compounds that bound at site II. In addition to binding at site II, 6 interacted at site I (warfarin binding site) as indicated by displacement of dansylamide and the shifting of NMR peaks of an HSA bound oleic acid molecule affected by warfarin site binding. Additional evidence for multiple site interaction was discovered when a percentage of 6 could be displaced by either ibuprofen or phenylbutazone. A competition assay was established using 6 to determine relative affinities of other antibacterial inhibitors for HSA.

Time-Resolved Transient Optical Absorption Study of Bis(terpyridyl)oligothiophenes and Their Metallo-Supramolecular Polymers with Zn(II) Ion Couplers.

PubMed

Rais, David; Menšík, Miroslav; Štenclová-Bláhová, Pavla; Svoboda, Jan; Vohlídal, Jiří; Pfleger, Jiří

2015-06-18

α,ω-Bis(terpyridyl)oligothiophenes spontaneously assemble with Zn(II) ions giving conjugated constitutional dynamic polymers (dynamers) of the metallo-supramolecular class, which potentially might be utilized in optoelectronics. Their photophysical properties, which are of great importance in this field of application, are strongly influenced by the dynamic morphology. It was assessed in this study by using ultrafast pump-probe optical absorption spectroscopy. We identified and characterized relaxation processes running in photoexcited molecules of these oligomers and dynamers and show impacts of disturbed coplanarity of adjacent rings (twisting the thiophene-thiophene and thiophene-terpyridyl bonds by attached hexyl side groups) and Zn(II) ion couplers on these processes. Major effects are seen in the time constants of rotational relaxation, intersystem crossing, and de-excitation lifetimes. The photoexcited states formed on different repeating units within the same dynamer chain do not interact with each other even at very high excitation density. The method is presented that allows determining the equilibrium fraction of unbound oligothiophene species in a dynamer solution, from which otherwise hardly accessible values of the average degree of polymerization of constitutionally dynamic chains in solution can be estimated.

Fluorescent indicator dyes for calcium ions

NASA Technical Reports Server (NTRS)

Grynkiewicz, Grzegorz (Inventor); Tsien, Roger Y. (Inventor)

1986-01-01

The present invention discloses a new class of highly fluorescent indicator dyes that are specific for calcium ions. The new fluorescent indicator dyes combine a stilbene-type fluorophore with a tetracarboxylate parent Ca.sup.2+ chelating compound having the octacoordinate pattern of liganding groups characteristic of EGTA and BAPTA. Preferred forms contain extra heterocyclic bridges to reinforce the ethylenic bond of the stilbene and to reduce hydrophobicity. Compared to their widely used predecessor, quin2, the new dyes offer up to thirty-fold brighter fluorescence, major changes in wavelength (not just intensity) upon Ca.sup.2+ binding, slightly lower affinities for Ca.sup.2+, slightly longer wavelengths of excitation, and considerably improved selectivity for Ca.sup.2+ over other divalent cations. These properties, particularly the wavelength sensitivity to Ca.sup.2+, make the dyes useful indicators for many intracellular applications, especially in single cells, adherent cell layers, or bulk tissues. The present invention also discloses an improved method for synthesizing alpha-acyloxyalkyl bromides wherein the bromides so synthesized are free of contaminating bis(1-bromoalkyl)ether. The improved method is exemplified herein in the synthesis of acetoxymethyl bromide, a compound useful in preparing the acetoxymethyl esters disclosed herein as novel Ca.sup.2+ specific fluorescent indicators.

Formation, characterization, aggregation, fluorescence and antioxidant properties of novel tetrasubstituted metal-free and metallophthalocyanines bearing (4-(methylthio)phenoxy) moieties

NASA Astrophysics Data System (ADS)

Yıldırım, Nurdan; Bilgiçli, Ahmet T.; Alici, Esma Hande; Arabacı, Gulnur; Yarasir, M. Nilüfer

2017-09-01

The synthesis and characterization of peripherally tetra 4-(methylthio)phenoxy substituted metal-free(2), Zn(II) (3) and Co(II) (4) phthalocyanine derivatives were reported. These newly synthesized phthalocyanine derivatives showed the enhanced solubility in organic solvents and they were characterized by a combination of elemental analysis, FTIR, 1H NMR, 13C NMR, UV-vis and MALDI-TOF/MS spectral data. Their aggregation properties were investigated in THF by UV-vis and fluorescence. These metal-free and metallophthalocyanine compounds were also evaluated for their total antioxidant abilities by using three different antioxidant methods such as 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging, ferrous ion chelating and reducing power activity. All tested compounds showed radical scavenging activity. The highest radical scavenging activity was found from cobalt phthalocyanine (4) compound respectively. IC50 values of the compounds and standards (BHT and Trolox) were also determined. The results showed that the compound 4 had the highest antioxidant activity among all tested compounds including standards. The tested phthalocyanine compounds had ferrous ion chelating activity. In addition, they showed very high reducing power. All tested compounds had higher reducing power than the standards such as ascorbic acid and BHT. The present study shows that the synthesized tetra phthalocyanine [M: 2H(2), Zn(II)(3), Co(II)(4)] with four peripheral 4-(methylthio) phenoxy compounds have the effective antioxidant properties that can be used as antioxidant agents.

Effects of metal on the biochemical properties of Helicobacter pylori HypB, a maturation factor of [NiFe]-hydrogenase and urease.

PubMed

Sydor, Andrew M; Liu, Jenny; Zamble, Deborah B

2011-03-01

The biosyntheses of the [NiFe]-hydrogenase and urease enzymes in Helicobacter pylori require several accessory proteins for proper construction of the nickel-containing metallocenters. The hydrogenase accessory proteins HypA and HypB, a GTPase, have been implicated in the nickel delivery steps of both enzymes. In this study, the metal-binding properties of H. pylori HypB were characterized, and the effects of metal binding on the biochemical behavior of the protein were examined. The protein can bind stoichiometric amounts of Zn(II) or Ni(II), each with nanomolar affinity. Mutation of Cys106 and His107, which are located between two major GTPase motifs, results in undetectable Ni(II) binding, and the Zn(II) affinity is weakened by 2 orders of magnitude. These two residues are also required for the metal-dependent dimerization observed in the presence of Ni(II) but not Zn(II). The addition of metals to the protein has distinct impacts on GTPase activity, with zinc significantly reducing GTP hydrolysis to below detectable levels and nickel only slightly altering the k(cat) and K(m) of the reaction. The regulation of HypB activities by metal binding may contribute to the maturation of the nickel-containing enzymes.

Quantitative evaluation of lectin-reactive glycoforms of α(1)-acid glycoprotein using lectin affinity capillary electrophoresis with fluorescence detection.

PubMed

Shimura, Kiyohito; Tamura, Mayumi; Toda, Tosifusa; Yazawa, Shin; Kasai, Ken-ichi

2011-08-01

α(1)-Acid glycoprotein (AGP) was previously shown to be a marker candidate of disease progression and prognosis of patients with malignancies by analysis of its glycoforms via lectins. Herein, affinity capillary electrophoresis of fluorescein-labeled AGP using lectins with the aid of laser-induced fluorescence detection was developed for quantitative evaluation of the fractional ratios of concanavalin A-reactive or Aleuria aurantia lectin-reactive AGP. Labeled AGP was applied at the anodic end of a fused-silica capillary (50 μm id, 360 μm od, 27 cm long) coated with linear polyacryloyl-β-alanyl-β-alanine, and electrophoresis was carried out for about 10 min in 60 mM 3-morpholinopropane-1-sulfonic acid-NaOH buffer (pH 7.35). Addition of the lectins to the anode buffer resulted in the separation of lectin-reactive glycoform peaks from lectin-non-reactive glycoform peaks. Quantification of the peak area of each group revealed that the percent of lectin-reactive AGP is independent of a labeling ratio ranging from 0.4 to 1.5 mol fluorescein/mol AGP, i.e. the standard deviation of 0.5% for an average of 59.9% (n=3). In combination with a facile procedure for micro-purification of AGP from serum, the present procedure, marking the reactivity of AGP with lectins, should be useful in determining the prognosis for a large number of patients with malignancies. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Dicyanovinylnaphthalenes for neuroimaging of amyloids and relationships of electronic structures and geometries to binding affinities

PubMed Central

Petrič, Andrej; Johnson, Scott A.; Pham, Hung V.; Li, Ying; Čeh, Simon; Golobič, Amalija; Agdeppa, Eric D.; Timbol, Gerald; Liu, Jie; Keum, Gyochang; Satyamurthy, Nagichettiar; Kepe, Vladimir; Houk, Kendall N.; Barrio, Jorge R.

2012-01-01

The positron-emission tomography (PET) probe 2-(1-[6-[(2-fluoroethyl)(methyl)amino]-2-naphthyl]ethylidene) (FDDNP) is used for the noninvasive brain imaging of amyloid-β (Aβ) and other amyloid aggregates present in Alzheimer’s disease and other neurodegenerative diseases. A series of FDDNP analogs has been synthesized and characterized using spectroscopic and computational methods. The binding affinities of these molecules have been measured experimentally and explained through the use of a computational model. The analogs were created by systematically modifying the donor and the acceptor sides of FDDNP to learn the structural requirements for optimal binding to Aβ aggregates. FDDNP and its analogs are neutral, environmentally sensitive, fluorescent molecules with high dipole moments, as evidenced by their spectroscopic properties and dipole moment calculations. The preferred solution-state conformation of these compounds is directly related to the binding affinities. The extreme cases were a nonplanar analog t-butyl-FDDNP, which shows low binding affinity for Aβ aggregates (520 nM Ki) in vitro and a nearly planar tricyclic analog cDDNP, which displayed the highest binding affinity (10 pM Ki). Using a previously published X-ray crystallographic model of 1,1-dicyano-2-[6-(dimethylamino)naphthalen-2-yl]propene (DDNP) bound to an amyloidogenic Aβ peptide model, we show that the binding affinity is inversely related to the distortion energy necessary to avoid steric clashes along the internal surface of the binding channel. PMID:23012452

New tris(dopamine) derivative as an iron chelator. Synthesis, solution thermodynamic stability, and antioxidant research.

PubMed

Zhang, Qingchun; Jin, Bo; Shi, Zhaotao; Wang, Xiaofang; Lei, Shan; Tang, Xingyan; Liang, Hua; Liu, Qiangqiang; Gong, Mei; Peng, Rufang

2017-06-01

A new tris(dopamine) derivative, containing three dopamine chelate moieties which were attached to a trimesic acid molecular scaffold, has been prepared and fully characterized by NMR, FTIR and HRMS. The solution thermodynamic stability of the chelator with Fe(III), Mg(II), Zn(II) and Fe(II) ions was investigated. Results demonstrated that the chelator exhibited effective binding ability and improved selectivity to Fe(III) ion. The chelator possessed affinity similar to that of diethylenetriaminepentaacetic acid chelator for Fe(III) ion. The high affinity could be attributed to the favorable geometric arrangement between the chelator and Fe(III) ion coordination preference. The chelator also exhibited high antioxidant activity and nontoxicity to neuron-like rat pheochromocytoma cells. Hence, the chelator could be used as chelating agent for iron overload situations without depleting essential metal ions, such as Mg(II) and Zn(II) ions. Copyright © 2017. Published by Elsevier Inc.

Crystal structure and ligand affinity of avidin in the complex with 4‧-hydroxyazobenzene-2-carboxylic acid

NASA Astrophysics Data System (ADS)

Strzelczyk, Paweł; Bujacz, Grzegorz

2016-04-01

Avidin is a protein found in egg white that binds numerous organic compounds with high affinity, especially biotin and its derivatives. Due to its extraordinary affinity for its ligands, avidin is extensively used in biotechnology. X-ray crystallography and fluorescence-based biophysical techniques were used to show that avidin binds the dye 4‧-hydroxyazobenzene-2-carboxylic acid (HABA) with a lower affinity than biotin. The apparent dissociation constant determined for the avidin complex with HABA by microscale thermophoresis (MST) is 4.12 μM. The crystal structure of avidin-HABA complex was determined at a resolution of 2.2 Å (PDB entry 5chk). The crystals belong to a hexagonal system, in the space group P6422. In that structure, the hydrazone tautomer of HABA is bound at the bottom part of the central calyx near the polar residues. We show interactions of the dye with avidin and compare them with the previously reported avidin-biotin complex.

Structural locus of transmucosal albumin efflux in canine ileum. A fluorescent study.

PubMed

Granger, D N; Cook, B H; Taylor, A E

1976-12-01

This study demonstrates the effects of elevated intestinal venous pressure on the intestinal tissue spaces and the histological locus of the transmucosal albumin flux under such conditions. The authors were able to localize albumin in the tissues using an Evans blue-albumin fluorescence technique. This technique makes use of the fluorescence properties and albumin affinity of Evans blue dye (T-1824). Evans blue dye has a high affinity for albumin and emits a red-orange fluorescence at a wavelength of 720 nm. Evans blue was mixed with a solution of bovine serum albumin at concentrations that yield negligible amounts of free dye. Control ileal samples were obtained in order to visualize the natural tissue morphology and fluorescence. The Evans blue-albumin solution was injected and tissue samples were obtained 15 and 60 min postinjection, then venous outflow was occluded and after 15 and 60 min the tissues were sampled. Each sample was immediately frozen, freeze dried, embedded in paraffin, and 7-mu sections were made. The Evans blue-albumin was demonstrated histologically with a fluorescence microscope. No leakage sites were apparent at normal venous pressures. However, after elevation of venous pressure, Evans blue-albumin was observed in the interepithelial and/or intraepithelial spaces of villus tips, but no Evans blue-albumin was observed either between or within the epithelial cells of the crypts, or within the tubular crypt lumina. These results indicate that at elevated venous pressures, the transmucosal albumin flux occurs exclusively at the villus tip region, suggesting a great vulnerability of the cells found in this region to elevations in tissue pressure as compared to the crypt epithelial cells.

Synthesis of a ratiometric fluorescent peptide sensor for the highly selective detection of Cd2+.

PubMed

Li, Yan; Li, Lianzhi; Pu, Xuewei; Ma, Guolin; Wang, Erqiong; Kong, Jinming; Liu, Zhipeng; Liu, Yangzhong

2012-06-15

A novel ratiometric fluorescent peptidyl chemosensor (Dansyl-Cys-Pro-Gly-Cys-Trp-NH(2), D-P5) for metal ions detection has been synthesized via Fmoc solid-phase peptide synthesis. The chemosensor exhibited a high selectivity for Cd(2+) over other metal ions including competitive transition and Group I and II metal ions in neutral pH. The fluorescence emission intensity of D-P5 was significantly enhanced in the presence of Cd(2+) by fluorescent resonance energy transfer (FRET) and chelation enhanced fluorescence (CHEF) effects. The binding stoichiometry, detection limit, binding affinity, reversibility and pH sensitivity of the sensor for Cd(2+) were investigated. Copyright © 2012 Elsevier Ltd. All rights reserved.

New heteroleptic Zn(II) complexes of thiosemicarbazone and diimine Co-Ligands: Structural analysis and their biological impacts

NASA Astrophysics Data System (ADS)

Mathan Kumar, Shanmugaiah; Kesavan, Mookkandi Palsamy; Vinoth Kumar, Gujuluva Gangatharan; Sankarganesh, Murugesan; Chakkaravarthi, Ganesan; Rajagopal, Gurusamy; Rajesh, Jegathalaprathaban

2018-02-01

A thiosemicarbazone ligand HL appended new Zn(II) complexes [Zn(L)(bpy)] (1) and [Zn(L)(phen)] (2) (where, HL = {2-(3-bromo-5-chloro-2-hydroxybenzylidene)-N-phenylhydrazinecarbothioamide}, bpy = 2, 2‧-bipyridine and phen = 1, 10-phenanthroline) have been synthesized and well characterized using conventional spectroscopic techniques viz.,1H NMR, FTIR and UV-Vis spectra. The crystal structures of complexes 1 and 2 have been determined by single crystal X-ray diffraction studies. Both the complex 1 (τ = 0.5) and 2 (τ = 0.37) possesses square based pyramidally distorted trigonal bipyramidal geometry. The ground state electronic structures of complexes 1 and 2 were investigated by DFT/B3LYP theoretical analysis using 6-311G (d,p) and LANL2DZ basis set level. The superior DNA binding ability of complex 2 has been evaluated using absorption and fluorescence spectral titration studies. Antimicrobial evaluation reveals that complex 2 endowed better screening than HL and complex 1 against both bacterial as well as fungal species. Consequently, complex 2 possesses highest antibacterial screening against Staphylococcus aureus (MIC = 3.0 ± 0.23 mM) and antifungal screening against Candida albicans (MIC = 6.0 ± 0.11 mM). Furthermore, the anticancer activity of the ligand HL, complexes 1 and 2 have been examined against the MCF-7 cell line (Human breast cancer cell line) using MTT assay. It is remarkable that complex 2 (12 ± 0.67 μM) show highest anticancer activity than HL (25.0 ± 0.91 μM) and complex 1 (15 ± 0.88 μM) due to the presence of phen ligand moiety.

Structural, spectroscopic and thermal characterization of 2-tert-butylaminomethylpyridine-6-carboxylic acid methylester and its Fe(III), Co(II), Ni(II), Cu(II), Zn(II) and UO(2)(II) complexes.

PubMed

Mohamed, Gehad G; El-Gamel, Nadia E A

2005-04-01

Fe(III), Co(II), Ni(II), Cu(II), Zn(II) and UO(2)(II) complexes with the ligand 2-tert-butylaminomethylpyridine-6-carboxylic acid methylester (HL(2)) have been prepared and characterized by elemental analyses, molar conductance, magnetic moment, thermal analysis and spectral data. 1:1 M:HL(2) complexes, with the general formula [M(HL(2))X(2)].nH(2)O (where M = Co(II) (X = Cl, n = 0), Ni(II) (X = Cl, n = 3), Cu(II) (grey colour, X = AcO, n = 1), Cu(II) (yellow colour, X = Cl, n = 0) and Zn(II) (X = Br, n = 0). In addition, the Fe(III) and UO(2)(II) complexes of the type 1:2 M:HL(2) and with the formulae [Fe(L(2))(2)]Cl and [UO(2)(HL(2))(2)](NO(3))(2) are prepared. From the IR data, it is seen that HL(2) ligand behaves as a terdentate ligand coordinated to the metal ions via the pyridyl N, carboxylate O and protonated NH group; except the Fe(III) complex, it coordinates via the deprotonated NH group. This is supported by the molar conductance data, which show that all the complexes are non-electrolytes, while the Fe(III) and UO(2)(II) complexes are 1:1 electrolytes. IR and H1-NMR spectral studies suggest a similar behaviour of the Zn(II) complex in solid and solution states. From the solid reflectance spectral data and magnetic moment measurements, the complexes have a trigonal bipyramidal (Co(II), Ni(II), Cu(II) and Zn(II) complexes) and octahedral (Fe(III), UO(2)(II) complexes) geometrical structures. The thermal behaviour of the complexes is studied and the different dynamic parameters are calculated applying Coats-Redfern equation.

Affinity in electrophoresis.

PubMed

Heegaard, Niels H H

2009-06-01

The journal Electrophoresis has greatly influenced my approaches to biomolecular affinity studies. The methods that I have chosen as my main tools to study interacting biomolecules--native gel and later capillary zone electrophoresis--have been the topic of numerous articles in Electrophoresis. Below, the role of the journal in the development and dissemination of these techniques and applications reviewed. Many exhaustive reviews on affinity electrophoresis and affinity CE have been published in the last few years and are not in any way replaced by the present deliberations that are focused on papers published by the journal.

Azilsartan and its Zn(II) complex. Synthesis, anticancer mechanisms of action and binding to bovine serum albumin.

PubMed

Martínez, Valeria R; Aguirre, María V; Todaro, Juan S; Piro, Oscar E; Echeverría, Gustavo A; Ferrer, Evelina G; Williams, Patricia A M

2018-04-01

Azilsartan is the eighth approved member of angiotensin II receptor blockers for hypertension treatment. Considering that some drugs have additional effects when administered, we studied its effects and mechanisms of action on a human lung cancer cell line A549. We have also modified the structure of the drug by complexation with Zn(II) cation and assayed the anticancer effect. The crystal structure of the new binuclear Zn(II) complex, for short [Zn 2 (azil) 2 (H 2 O) 4 ]·2H 2 O (ZnAzil), was determined by X-ray diffraction methods. The zinc ions are bridged by azilsartan ligands through their carboxylate oxygen and oxadiazol nitrogen atoms. The compounds were examined for their cytotoxic effects against human lung fibroblast (MRC5) and human lung cancer (A549) cell lines. Azilsartan displayed low cytotoxic effects at 150 μM concentrations in A549 human lung cancer cells but the higher effect measured for the Zn complex suggested that this compound may act as an anticancer agent. An apoptotic oxidative stress mechanism of action via the mitochondrial-dependent intrinsic pathway has been determined. Besides, the compounds exerted weak cytotoxic effects in the normal lung related cell line MRC5. Binding constants of the complex formed between each compound and bovine serum albumin (BSA) are in the intermediate range, hence suggesting that azilsartan and ZnAzil could be bonded and transported by BSA. Copyright © 2018 Elsevier Ltd. All rights reserved.

Folic acid-targeted magnetic Tb-doped CeF3 fluorescent nanoparticles as bimodal probes for cellular fluorescence and magnetic resonance imaging.

PubMed

Ma, Zhi-Ya; Liu, Yu-Ping; Bai, Ling-Yu; An, Jie; Zhang, Lin; Xuan, Yang; Zhang, Xiao-Shuai; Zhao, Yuan-Di

2015-10-07

Magnetic fluorescent nanoparticles (NPs) have great potential applications for diagnostics, imaging and therapy. We developed a facile polyol method to synthesize multifunctional Fe3O4@CeF3:Tb@CeF3 NPs with small size (<20 nm), high water solubility and good biocompatibility. The NPs were modified by ligand exchange reactions with citric acid (CA) to obtain carboxyl-functionalized NPs (Fe3O4@CeF3:Tb@CeF3-COOH). Folic acid (FA) as an affinity ligand was then covalently conjugated onto NPs to yield Fe3O4@CeF3:Tb@CeF3-FA NPs. They were then applied as multimodal imaging agents for simultaneous in vitro targeted fluorescence imaging and magnetic resonance imaging (MRI) of HeLa cells with overexpressed folate receptors (FR). The results indicated that these NPs had strong luminescence and enhanced T2-weighted MR contrast and would be promising candidates as multimodal probes for both fluorescence and MRI imaging.

Dimeric fluorescent energy transfer dyes comprising asymmetric cyanine azole-indolenine chromophores

DOEpatents

Glazer, Alexander N.; Benson, Scott C.

1996-01-01

Novel fluorescent DNA-staining dyes are provided combining asymmetric cyanine azole-indolenine dyes, which provide for strong DNA affinity, large Stokes shifts and emission in the red region of the spectrum. The dyes find particular application in gel electrophoresis and for labels which may be bound to a variety of compositions in a variety of contexts.

Design and Application of Synthetic Receptors for Recognition of Methylated Lysine and Supramolecular Affinity Labeling

NASA Astrophysics Data System (ADS)

Gober, Isaiah Nathaniel

This dissertation involves the design and synthesis of new synthetic receptors and their application in the molecular recognition of methylated lysine and their use as tools for chemical biology. The dissertation is divided into four parts. The first section focuses on the development of a novel labeling method that is based on ligand-directed affinity labeling principles. In this labeling method, a synthetic receptor that binds to trimethyl lysine (Kme3) is attached through a linker to an electrophilic tag group that can react with a nucleophilic amine in a histone peptide. This affinity labeling probe, which we called CX4-ONBD, is equipped with an electrophilic tag that allows for turn-on fluorescence labeling of Kme3 histone peitdes. We show that the probe gives a pronounced turn-on fluorescence response when it is incubated with a histone peptide that contains Kme3 and a nearby reactive lysine. This probe also displays >5-fold selectivity in covalent labeling over an unmethylated lysine peptide. This represents the first time a synthetic receptor has been used for affinity labeling purposes, and it also expands on the chemical toolkit that is available for sensing PTMs like lysine methylation. In the second section, the supramolecular affinity labeling method that was optimized using CX4-ONBD was applied to the development of a real-time assay for measuring enzymatic activity. More specifically, the probe was used to create a turn-on fluorescence assay for histone deacetylase (HDAC) activity and for inhibitor screening and IC50 determination. Most commercial kits for HDAC activity have limited substrate scope, and other common methods used for characterizing enzymatic activity often require chromatographic separation and are therefore not high-throughput. This small molecule receptor-mediated affinity labeling strategy allowed for facile readout of HDAC activity and inhibition. Overall, this application of supramolecular affinity labeling expands on the

Fluorescent sensors for activity and regulation of the nitrate transceptor CHL1/NRT1.1 and oligopeptide transporters

PubMed Central

Ho, Cheng-Hsun; Frommer, Wolf B

2014-01-01

To monitor nitrate and peptide transport activity in vivo, we converted the dual-affinity nitrate transceptor CHL1/NRT1.1/NPF6.3 and four related oligopeptide transporters PTR1, 2, 4, and 5 into fluorescence activity sensors (NiTrac1, PepTrac). Substrate addition to yeast expressing transporter fusions with yellow fluorescent protein and mCerulean triggered substrate-dependent donor quenching or resonance energy transfer. Fluorescence changes were nitrate/peptide-specific, respectively. Like CHL1, NiTrac1 had biphasic kinetics. Mutation of T101A eliminated high-affinity transport and blocked the fluorescence response to low nitrate. NiTrac was used for characterizing side chains considered important for substrate interaction, proton coupling, and regulation. We observed a striking correlation between transport activity and sensor output. Coexpression of NiTrac with known calcineurin-like proteins (CBL1, 9; CIPK23) and candidates identified in an interactome screen (CBL1, KT2, WNKinase 8) blocked NiTrac1 responses, demonstrating the suitability for in vivo analysis of activity and regulation. The new technology is applicable in plant and medical research. DOI: http://dx.doi.org/10.7554/eLife.01917.001 PMID:24623305

Identification of a Novel Indoline Derivative for in Vivo Fluorescent Imaging of Blood-Brain Barrier Disruption in Animal Models

PubMed Central

2013-01-01

Disruption of the blood-brain barrier (BBB) can occur in various pathophysiological conditions. Administration of extraneous tracers that can pass the disrupted, but not the intact, BBB and detection of the extravasation have been widely used to assess BBB disruption in animal models. Although several fluorescent tracers have been successfully used, the administration of these tracers basically requires intravascular injection, which can be laborious when using small animals such as zebrafish. To identify fluorescent tracers that could be easily administered into various animal models and visualize the BBB disruption in vivo, we prepared nine structurally related indoline derivatives (IDs) as a minimum set of diverse fluorescent compounds. We found that one ID, ZMB741, had the highest affinity for serum albumin and emitted the strongest fluorescence in the presence of serum albumin of the nine IDs tested. The affinity to serum albumin and the fluorescence intensity was superior to those of Evans blue and indocyanine green that have been conventionally used to assess the BBB disruption. We showed that ZMB741 could be administered into zebrafish by static immersion or mice by intraperitoneal injection and visualizes the active disruption of their BBB. These results suggest that ZMB741 can be a convenient and versatile tool for in vivo fluorescent imaging of BBB disruption in various animal models. The strategy used in this study can also be applied to diversity-oriented libraries to identify novel fluorescent tracers that may be superior to ZMB741. PMID:23668665

Adjoint affine fusion and tadpoles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Urichuk, Andrew, E-mail: andrew.urichuk@uleth.ca; Walton, Mark A., E-mail: walton@uleth.ca; International School for Advanced Studies

2016-06-15

We study affine fusion with the adjoint representation. For simple Lie algebras, elementary and universal formulas determine the decomposition of a tensor product of an integrable highest-weight representation with the adjoint representation. Using the (refined) affine depth rule, we prove that equally striking results apply to adjoint affine fusion. For diagonal fusion, a coefficient equals the number of nonzero Dynkin labels of the relevant affine highest weight, minus 1. A nice lattice-polytope interpretation follows and allows the straightforward calculation of the genus-1 1-point adjoint Verlinde dimension, the adjoint affine fusion tadpole. Explicit formulas, (piecewise) polynomial in the level, are writtenmore » for the adjoint tadpoles of all classical Lie algebras. We show that off-diagonal adjoint affine fusion is obtained from the corresponding tensor product by simply dropping non-dominant representations.« less

Broad host range vectors for expression of proteins with (Twin-) Strep-tag, His-tag and engineered, export optimized yellow fluorescent protein

PubMed Central

2013-01-01

Background In current protein research, a limitation still is the production of active recombinant proteins or native protein associations to assess their function. Especially the localization and analysis of protein-complexes or the identification of modifications and small molecule interaction partners by co-purification experiments requires a controllable expression of affinity- and/or fluorescence tagged variants of a protein of interest in its native cellular background. Advantages of periplasmic and/or homologous expressions can frequently not be realized due to a lack of suitable tools. Instead, experiments are often limited to the heterologous production in one of the few well established expression strains. Results Here, we introduce a series of new RK2 based broad host range expression plasmids for inducible production of affinity- and fluorescence tagged proteins in the cytoplasm and periplasm of a wide range of Gram negative hosts which are designed to match the recently suggested modular Standard European Vector Architecture and database. The vectors are equipped with a yellow fluorescent protein variant which is engineered to fold and brightly fluoresce in the bacterial periplasm following Sec-mediated export, as shown from fractionation and imaging studies. Expression of Strep-tag®II and Twin-Strep-tag® fusion proteins in Pseudomonas putida KT2440 is demonstrated for various ORFs. Conclusion The broad host range constructs we have produced enable good and controlled expression of affinity tagged protein variants for single-step purification and qualify for complex co-purification experiments. Periplasmic export variants enable production of affinity tagged proteins and generation of fusion proteins with a novel engineered Aequorea-based yellow fluorescent reporter protein variant with activity in the periplasm of the tested Gram-negative model bacteria Pseudomonas putida KT2440 and Escherichia coli K12 for production, localization or co

In vivo fluorescence imaging of hepatocellular carcinoma using a novel GPC3-specific aptamer probe

PubMed Central

Zhao, Menglong; Dong, Lili; Liu, Zhuang; Yang, Shuohui

2018-01-01

Background Glypican-3 (GPC3) is highly expressed in most of the hepatocellular carcinomas (HCCs), even in small HCCs. It may be used as a potential biomarker for early detection of HCC. The aptamer is a promising targeting agent with unique advantages over antibody. This study was to introduce a novel GPC3 specific aptamer (AP613-1), to verify its specific binding property in vitro, and to evaluate its targeting efficiency in vivo by performing near-infrared (NIR) fluorescence imaging on an HCC xenograft model. Methods AP613-1 was generated from the systematic evolution of ligands by exponential enrichment. Flow cytometry and aptamer-based immunofluorescence imaging were performed to verify the binding affinity of AP613-1 to GPC3 in vitro. NIR Fluorescence images of nude mice with unilateral (n=12) and bilateral (n=4) subcutaneous xenograft tumors were obtained. Correlation between the tumor fluorescence intensities in vivo and ex vivo was analyzed. Results AP613-1 could specifically bind to GPC3 in vitro. In vivo and ex vivo tumors, fluorescence intensities were in excellent correlation (P<0.001, r=0.968). The fluorescence intensity is significantly higher in tumors given Alexa Fluor 750 (AF750) labeled AP613-1 than in those given AF750 labeled initial ssDNA library both in vivo (P<0.001) and ex vivo (P=0.022). In the mice with bilateral subcutaneous tumors injected with AF750 labeled AP613-1, Huh-7 tumors showed significantly higher fluorescence intensities than A549 tumors both in vivo (P=0.016) and ex vivo (P=0.004). Conclusions AP613-1 displays a specific binding affinity to GPC3 positive HCC. Fluorescently labeled AP613-1 could be used as an imaging probe to subcutaneous HCC in xenograft models. PMID:29675356

Fluorescent and Lanthanide Labeling for Ligand Screens, Assays, and Imaging

PubMed Central

Josan, Jatinder S.; De Silva, Channa R.; Yoo, Byunghee; Lynch, Ronald M.; Pagel, Mark D.; Vagner, Josef; Hruby, Victor J.

2012-01-01

The use of fluorescent (or luminescent) and metal contrast agents in high-throughput screens, in vitro assays, and molecular imaging procedures has rapidly expanded in recent years. Here we describe the development and utility of high-affinity ligands for cancer theranostics and other in vitro screening studies. In this context, we also illustrate the syntheses and use of heteromultivalent ligands as targeted imaging agents. PMID:21318902

The high affinity of small-molecule antioxidants for hemoglobin.

PubMed

Puscas, Cristina; Radu, Luana; Carrascoza, Francisco; Mot, Augustin C; Amariei, Diana; Lungu, Oana; Scurtu, Florina; Podea, Paula; Septelean, Raluca; Matei, Alina; Mic, Mihaela; Attia, Amr A; Silaghi-Dumitrescu, Radu

2018-06-18

Hemoglobin has previously been shown to display ascorbate peroxidase and urate peroxidase activity, with measurable Michaelis-Menten parameters that reveal a particularly low Km for ascorbate as well as for urate - lower than the respective in vivo concentrations of these antioxidants in blood. Also, direct detection of a hemoglobin-ascorbate interaction was possible by monitoring the 1H-NMR spectrum of ascorbate in the presence of hemoglobin. The relative difference in structures between ascorbate and urate may raise the question as to exactly what the defining structural features would be, for a substrate that binds to hemoglobin with high affinity. Reported here are Michaelis-Menten parameters for hemoglobin acting as peroxidase against a number of other substrates of varying structures - gallate, caffeate, rutin, 3-hydroxyflavone, 3,6-dihydroxyflavone, quercetin, epicatechin, luteolin - all with high affinities (some higher than those of physiologically-relevant redox partners of Hb - ascorbate and urate). Moreover, this high affinity appears general to animal hemoglobins. 1 H-NMR and 13 C-NMR spectra reveal a general pattern wherein small hydrophilic antioxidants appear to all have their signals affected, presumably due to binding to hemoglobin. Fluorescence and calorimetry measurements confirm these conclusions. Docking calculations confirm the existence of binding sites on hemoglobin and on myoglobin for ascorbate as well as for other antioxidants. Support is found for involvement of Tyr42 in binding of three out of the four substrates investigated in the case of hemoglobin (including ascorbate and urate, as blood-contained relevant substrates), but also for Tyr145 (with urate and caffeate) and Tyr35 (with gallate). Copyright © 2018 Elsevier Inc. All rights reserved.

Non-affine deformations in polymer hydrogels

PubMed Central

Wen, Qi; Basu, Anindita; Janmey, Paul A.; Yodh, A. G.

2012-01-01

Most theories of soft matter elasticity assume that the local strain in a sample after deformation is identical everywhere and equal to the macroscopic strain, or equivalently that the deformation is affine. We discuss the elasticity of hydrogels of crosslinked polymers with special attention to affine and non-affine theories of elasticity. Experimental procedures to measure non-affine deformations are also described. Entropic theories, which account for gel elasticity based on stretching out individual polymer chains, predict affine deformations. In contrast, simulations of network deformation that result in bending of the stiff constituent filaments generally predict non-affine behavior. Results from experiments show significant non-affine deformation in hydrogels even when they are formed by flexible polymers for which bending would appear to be negligible compared to stretching. However, this finding is not necessarily an experimental proof of the non-affine model for elasticity. We emphasize the insights gained from experiments using confocal rheoscope and show that, in addition to filament bending, sample micro-inhomogeneity can be a significant alternative source of non-affine deformation. PMID:23002395

Experimental and theoretical analysis of a rare nitrato bridged 3d-4f complex containing LaZn2 core synthesized from a Zn(II) metalloligand

NASA Astrophysics Data System (ADS)

Sreejith, S. S.; Mohan, Nithya; Kurup, M. R. Prathapachandra

2018-02-01

A trinulcear Zn2La Schiff base complex was synthesized using slow-solvent evaporation technique from a Zn(II) mononuclear metalloligand by 2:1 addition with La(NO3)3 salt. Single crystal XRD analysis revealed a rare nitrato bridged trinuclear entity which is seldom seen in these class of ligand systems. Qualitative and quantitative analysis of intermolecular interactions/short contacts were done using Hirshfeld surface and 2D finger print analysis. The thermally stable, blue luminescent compound exhibits internal heavy atom effect thereby quenching the emission intensity of the ligand. DFT calculations were performed on the compound to analyze frontier orbitals and also ESP plots were used to monitor nucleophilic/electrophilic regions on the compound and its implications on hydrogen bonding. A comparison of the bond orders and atomic charges on the trinuclear compound and the Zn(II) metalloligand precursor was performed to substantiate the formation of the trinuclear product through ligand exchange.

Simultaneous cryo X-ray ptychographic and fluorescence microscopy of green algae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deng, Junjing; Vine, David J.; Chen, Si

Trace metals play important roles in normal and in disease-causing biological functions. X-ray fluorescence microscopy reveals trace elements with no dependence on binding affinities (unlike with visible light fluorophores) and with improved sensitivity relative to electron probes. However, X-ray fluorescence is not very sensitive for showing the light elements that comprise the majority of cellular material. Here we show that X-ray ptychography can be combined with fluorescence to image both cellular structure and trace element distribution in frozen-hydrated cells at cryogenic temperatures, with high structural and chemical fidelity. Ptychographic reconstruction algorithms deliver phase and absorption contrast images at a resolutionmore » beyond that of the illuminating lens or beam size. Using 5.2-keV X-rays, we have obtained sub-30-nm resolution structural images and ~90-nm-resolution fluorescence images of several elements in frozen-hydrated green algae. Finally, this combined approach offers a way to study the role of trace elements in their structural context.« less

Simultaneous cryo X-ray ptychographic and fluorescence microscopy of green algae

DOE PAGES

Deng, Junjing; Vine, David J.; Chen, Si; ...

2015-02-24

Trace metals play important roles in normal and in disease-causing biological functions. X-ray fluorescence microscopy reveals trace elements with no dependence on binding affinities (unlike with visible light fluorophores) and with improved sensitivity relative to electron probes. However, X-ray fluorescence is not very sensitive for showing the light elements that comprise the majority of cellular material. Here we show that X-ray ptychography can be combined with fluorescence to image both cellular structure and trace element distribution in frozen-hydrated cells at cryogenic temperatures, with high structural and chemical fidelity. Ptychographic reconstruction algorithms deliver phase and absorption contrast images at a resolutionmore » beyond that of the illuminating lens or beam size. Using 5.2-keV X-rays, we have obtained sub-30-nm resolution structural images and ~90-nm-resolution fluorescence images of several elements in frozen-hydrated green algae. Finally, this combined approach offers a way to study the role of trace elements in their structural context.« less

Fluorescence spectroscopy approaches for the development of a real-time organophosphate detection system using an enzymatic sensor.

PubMed

Carullo, Paola; Cetrangolo, Giovanni Paolo; Mandrich, Luigi; Manco, Giuseppe; Febbraio, Ferdinando

2015-02-09

Organophosphates are organic substances that contain a phosphoryl or a thiophosphoryl bond. They are mainly used around the world as pesticides, but can also be used as chemical warfare agents. Their detection is normally entrusted to techniques like GC- and LC-MS that, although sensitive, do not allow their identification on site and in real time. We have approached their identification by exploiting the high-affinity binding of these compounds with the esterase 2 from Alicyclobacillus acidocaldarius. Using an in silico analysis to evaluate the binding affinities of the enzyme with organophosphate inhibitors, like paraoxon, and other organophosphate compounds, like parathion, chlorpyriphos, and other organophosphate thio-derivatives, we have designed fluorescence spectroscopy experiments to study the quenching of the tryptophan residues after esterase 2 binding with the organophosphate pesticides. The changes in the fluorescence signals permitted an immediate and quantitative identification of these compounds from nano- to picomolar concentrations. A fluorescence based polarity-sensitive probe (ANS) was also employed as a means to understand the extent of the interactions involved, as well as to explore other ways to detect organophosphate pesticides. Finally, we designed a framework for the development of a biosensor that exploits fluorescence technology in combination with a sensitive and very stable bio-receptor.

Lp-mixed affine surface area

NASA Astrophysics Data System (ADS)

Wang, Weidong; Leng, Gangsong

2007-11-01

According to the three notions of mixed affine surface area, Lp-affine surface area and Lp-mixed affine surface area proposed by Lutwak, in this article, we give the concept of ith Lp-mixed affine surface area such that the first and second notions of Lutwak are its special cases. Further, some Lutwak's results are extended associated with this concept. Besides, applying this concept, we establish an inequality for the volumes and dual quermassintegrals of a class of star bodies.

Modulating Uranium Binding Affinity in Engineered Calmodulin EF-Hand Peptides: Effect of Phosphorylation

PubMed Central

Pardoux, Romain; Sauge-Merle, Sandrine; Lemaire, David; Delangle, Pascale; Guilloreau, Luc; Adriano, Jean-Marc; Berthomieu, Catherine

2012-01-01

To improve our understanding of uranium toxicity, the determinants of uranyl affinity in proteins must be better characterized. In this work, we analyzed the contribution of a phosphoryl group on uranium binding affinity in a protein binding site, using the site 1 EF-hand motif of calmodulin. The recombinant domain 1 of calmodulin from A. thaliana was engineered to impair metal binding at site 2 and was used as a structured template. Threonine at position 9 of the loop was phosphorylated in vitro, using the recombinant catalytic subunit of protein kinase CK2. Hence, the T9TKE12 sequence was substituted by the CK2 recognition sequence TAAE. A tyrosine was introduced at position 7, so that uranyl and calcium binding affinities could be determined by following tyrosine fluorescence. Phosphorylation was characterized by ESI-MS spectrometry, and the phosphorylated peptide was purified to homogeneity using ion-exchange chromatography. The binding constants for uranyl were determined by competition experiments with iminodiacetate. At pH 6, phosphorylation increased the affinity for uranyl by a factor of ∼5, from Kd = 25±6 nM to Kd = 5±1 nM. The phosphorylated peptide exhibited a much larger affinity at pH 7, with a dissociation constant in the subnanomolar range (Kd = 0.25±0.06 nM). FTIR analyses showed that the phosphothreonine side chain is partly protonated at pH 6, while it is fully deprotonated at pH 7. Moreover, formation of the uranyl-peptide complex at pH 7 resulted in significant frequency shifts of the νas(P-O) and νs(P-O) IR modes of phosphothreonine, supporting its direct interaction with uranyl. Accordingly, a bathochromic shift in νas(UO2)2+ vibration (from 923 cm−1 to 908 cm−1) was observed upon uranyl coordination to the phosphorylated peptide. Together, our data demonstrate that the phosphoryl group plays a determining role in uranyl binding affinity to proteins at physiological pH. PMID:22870263

Dimeric fluorescent energy transfer dyes comprising asymmetric cyanine azole-indolenine chromophores

DOEpatents

Glazer, Alexander N.; Benson, Scott C.

1998-01-01

Novel fluorescent heterodimeric DNA-staining energy transfer dyes are provided combining asymmetric cyanine azole-indolenine dyes, which provide for strong DNA affinity, large Stokes shifts and emission in the red region of the spectrum. The dyes find particular application in gel electrophoresis and for labels which may be bound to a variety of compositions in a variety of contexts. Kits and individual compounds are provided, where the kits find use for simultaneous detection of a variety of moieties, particularly using a single narrow wavelength irradiation source. The individual compounds are characterized by high donor quenching and high affinity to dsDNA as a result of optimizing the length of the linking group separating the two chromophores.

Evaluation of immobilized metal-ion affinity chromatography (IMAC) as a technique for IgG(1) monoclonal antibodies purification: the effect of chelating ligand and support.

PubMed

Bresolin, I T L; Borsoi-Ribeiro, M; Tamashiro, W M S C; Augusto, E F P; Vijayalakshmi, M A; Bueno, S M A

2010-04-01

Monoclonal antibodies (MAbs) have been used for therapies and some analytical procedures as highly purified molecules. Many techniques have been applied and studied, focusing on monoclonal antibodies purification. In this study, an immobilized metal affinity chromatography membrane was developed and evaluated for the purification of anti-TNP IgG(1) mouse MAbs from cell culture supernatant after precipitation with a 50% saturated ammonium sulfate solution. The chelating ligands iminodiacetic acid, carboxymethylated aspartic acid (CM-Asp), nitrilotriacetic acid, and tris (carboxymethyl) ethylenediamine in agarose gels with immobilized Ni(II) and Zn(II) ions were compared for the adsorption and desorption of MAbs. The most promising chelating ligand--CM-Asp--was then coupled to poly(ethylene vinyl alcohol) (PEVA) hollow fiber membranes. According to SDS-PAGE and ELISA analyses, a higher selectivity and a purification factor of 85.9 (fraction eluted at 500 mM Tris) were obtained for IgG(1) using PEVA-CM-Asp-Zn(II). The anti-TNP MAb could be eluted under mild pH conditions causing no loss of antigen binding capacity.

A highly selective colorimetric and ratiometric fluorescent chemodosimeter for detection of fluoride ions based on 1,8-naphthalimide derivatives.

PubMed

Kai, Yumei; Hu, Yonghong; Wang, Kai; Zhi, Wenbiao; Liang, Mengmeng; Yang, Wenge

2014-01-24

A high selective colorimetric and ratiometric fluorescent probe based on 4-hydroxy-1, 8-naphthalimide was designed and synthesized to detect fluoride ions (F(-)). The sensing behavior of this probe was studied by UV-visible and fluorescence spectroscopy. The probe displays an 110 nm red-shift of fluorescence emission and the color changes from colorless to yellow by virtue of the strong affinity of F(-) toward silicon which can act as a new visual sensor for F(-). Copyright © 2013 Elsevier B.V. All rights reserved.

Interaction of fluorescent sensor with superparamagnetic iron oxide nanoparticles.

PubMed

Karunakaran, Chockalingam; Jayabharathi, Jayaraman; Sathishkumar, Ramalingam; Jayamoorthy, Karunamoorthy

2013-06-01

To sense superparamagnetic iron oxides (Fe2O3 and Fe3O4) nanocrystals a sensitive bioactive phenanthroimidazole based fluorescent molecule, 2-(4-fluorophenyl)-1-phenyl-1H-phenanthro [9,10-d] imidazole has been designed and synthesized. Electronic spectral studies show that phenanthroimidazole is bound to the surface of iron oxide semiconductors. Fluorescent enhancement has been explained on the basis of photo-induced electron transfer (PET) mechanism and apparent binding constants have been deduced. Binding of phenanthroimidazole with iron oxide nanoparticles lowers the HOMO and LUMO energy levels of phenanthroimidazole molecule. Chemical affinity between the nitrogen atom of the phenanthroimidazole and Fe(2+) and Fe(3+) ions on the surface of the nano-oxide may result in strong binding of the phenanthroimidazole derivative with the nanoparticles. The electron injection from the photoexcited phenanthroimidazole to the iron oxides conduction band explains the enhanced fluorescence. Copyright © 2013 Elsevier B.V. All rights reserved.

BODIPY-Based Fluorescent Probes for Sensing Protein Surface-Hydrophobicity.

PubMed

Dorh, Nethaniah; Zhu, Shilei; Dhungana, Kamal B; Pati, Ranjit; Luo, Fen-Tair; Liu, Haiying; Tiwari, Ashutosh

2015-12-18

Mapping surface hydrophobic interactions in proteins is key to understanding molecular recognition, biological functions, and is central to many protein misfolding diseases. Herein, we report synthesis and application of new BODIPY-based hydrophobic sensors (HPsensors) that are stable and highly fluorescent for pH values ranging from 7.0 to 9.0. Surface hydrophobic measurements of proteins (BSA, apomyoglobin, and myoglobin) by these HPsensors display much stronger signal compared to 8-anilino-1-naphthalene sulfonic acid (ANS), a commonly used hydrophobic probe; HPsensors show a 10- to 60-fold increase in signal strength for the BSA protein with affinity in the nanomolar range. This suggests that these HPsensors can be used as a sensitive indicator of protein surface hydrophobicity. A first principle approach is used to identify the molecular level mechanism for the substantial increase in the fluorescence signal strength. Our results show that conformational change and increased molecular rigidity of the dye due to its hydrophobic interaction with protein lead to fluorescence enhancement.

Identification and Characterization of a High-Affinity Choline Uptake System of Brucella abortus

PubMed Central

Herrmann, Claudia K.; Bukata, Lucas; Melli, Luciano; Marchesini, M. Ines; Caramelo, Julio J.

2013-01-01

Phosphatidylcholine (PC), a common phospholipid of the eukaryotic cell membrane, is present in the cell envelope of the intracellular pathogen Brucella abortus, the etiological agent of bovine brucellosis. In this pathogen, the biosynthesis of PC proceeds mainly through the phosphatidylcholine synthase pathway; hence, it relies on the presence of choline in the milieu. These observations imply that B. abortus encodes an as-yet-unknown choline uptake system. Taking advantage of the requirement of choline uptake for PC synthesis, we devised a method that allowed us to identify a homologue of ChoX, the high-affinity periplasmic binding protein of the ABC transporter ChoXWV. Disruption of the choX gene completely abrogated PC synthesis at low choline concentrations in the medium, thus indicating that it is a high-affinity transporter needed for PC synthesis via the PC synthase (PCS) pathway. However, the synthesis of PC was restored when the mutant was incubated in media with higher choline concentrations, suggesting the presence of an alternative low-affinity choline uptake activity. By means of a fluorescence-based equilibrium-binding assay and using the kinetics of radiolabeled choline uptake, we show that ChoX binds choline with an extremely high affinity, and we also demonstrate that its activity is inhibited by increasing choline concentrations. Cell infection assays indicate that ChoX activity is required during the first phase of B. abortus intracellular traffic, suggesting that choline concentrations in the early and intermediate Brucella-containing vacuoles are limited. Altogether, these results suggest that choline transport and PC synthesis are strictly regulated in B. abortus. PMID:23161032

High affinity binding of amyloid β-peptide to calmodulin: Structural and functional implications.

PubMed

Corbacho, Isaac; Berrocal, María; Török, Katalin; Mata, Ana M; Gutierrez-Merino, Carlos

2017-05-13

Amyloid β-peptides (Aβ) are a major hallmark of Alzheimer's disease (AD) and their neurotoxicity develop with cytosolic calcium dysregulation. On the other hand, calmodulin (CaM), a protein which plays a major multifunctional role in neuronal calcium signaling, has been shown to be involved in the regulation of non-amyloidogenic processing of amyloid β precursor protein (APP). Using fluorescent 6-bromoacetyl-2-dimethylaminonaphthalene derivatives of CaM, Badan-CaM, and human amyloid β(1-42) HiLyte™-Fluor555, we show in this work that Aβ binds with high affinity to CaM through the neurotoxic Aβ25-35 domain. In addition, the affinity of Aβ for calcium-saturated CaM conformation is approximately 20-fold higher than for CaM conformation in the absence of calcium (apo-CaM). Moreover, the value of K d of 0.98 ± 0.11 nM obtained for Aβ1-42 dissociation from CaM saturated by calcium points out that CaM is one of the cellular targets with highest affinity for neurotoxic Aβ peptides. A major functional consequence of Aβ-CaM interaction is that it slowdowns Aβ fibrillation. The novel and high affinity interaction between calmodulin and Aβ shown in this work opens a yet-unexplored gateway to further understand the neurotoxic effect of Aβ in different neural cells and also to address the potential of calmodulin and calmodulin-derived peptides as therapeutic agents in AD. Copyright © 2017 Elsevier Inc. All rights reserved.

Tolerance of a knotted near infrared fluorescent protein to random circular permutation

PubMed Central

Pandey, Naresh; Kuypers, Brianna E.; Nassif, Barbara; Thomas, Emily E.; Alnahhas, Razan N.; Segatori, Laura; Silberg, Jonathan J.

2016-01-01

Bacteriophytochrome photoreceptors (BphP) are knotted proteins that have been developed as near-infrared fluorescent protein (iRFP) reporters of gene expression. To explore how rearrangements in the peptides that interlace into the knot within the BphP photosensory core affect folding, we subjected iRFP to random circular permutation using an improved transposase mutagenesis strategy and screened for variants that fluoresce. We identified twenty seven circularly permuted iRFP that display biliverdin-dependent fluorescence in Escherichia coli. The variants with the brightest whole cell fluorescence initiated translation at residues near the domain linker and knot tails, although fluorescent variants were discovered that initiated translation within the PAS and GAF domains. Circularly permuted iRFP retained sufficient cofactor affinity to fluoresce in tissue culture without the addition of biliverdin, and one variant displayed enhanced fluorescence when expressed in bacteria and tissue culture. This variant displayed a similar quantum yield as iRFP, but exhibited increased resistance to chemical denaturation, suggesting that the observed signal increase arose from more efficient protein maturation. These results show how the contact order of a knotted BphP can be altered without disrupting chromophore binding and fluorescence, an important step towards the creation of near-infrared biosensors with expanded chemical-sensing functions for in vivo imaging. PMID:27304983

Switching on fluorescence for selective visual recognition of naringenin and morin with a metal-organic coordination polymer of Zn(bix) [bix = 1,4-bis(imidazol-1-ylmethyl)benzene

NASA Astrophysics Data System (ADS)

Zhao, Xi Juan; Wang, Hui Juan; Liang, Li Jiao; Li, Yuan Fang

2013-02-01

Flavonoids such as naringenin and morin are ubiquitous in a wide range of foods isolated from plants, and have diverse effects on plants even on human health. Here, we establish a selective visual method for recognition of aringenin and morin based on the "switched on" fluorescence induced by a metal-organic coordination polymer of Zn(bix) [bix = 1,4-bis(imidazol-1-ylmethyl)benzene]. Owing to the coordination interaction of aringenin and morin with Zn(II) from the polymeric structure of Zn(bix), the conformational free rotation of naringenin and morin is restricted leading to relatively rigid structures. And as a consequence, the fluorescence is switched on. While luteolin and quercetin, holding a very similar structure with naringenin and morin, have no such fluorescence enhancement most likely owing to the 3'-hydroxy substitution in the B ring. Under 365 nm UV lamp light, we can visually recognize and discriminate naringenin and morin from them each other and luteolin as well as quercetin based on the colors of their emission. With this recognition system, the detection of naringenin and morin in human urine was made with satisfactory results.

Interaction of phenolic acids and their derivatives with human serum albumin: Structure-affinity relationships and effects on antioxidant activity.

PubMed

Zhang, Yunyue; Wu, Simin; Qin, Yinghui; Liu, Jiaxin; Liu, Jingwen; Wang, Qingyu; Ren, Fazheng; Zhang, Hao

2018-02-01

In this study, 111 phenolic acids and their derivatives were chosen to investigate their structure-affinity relationships when binding to human serum albumin (HSA), and effects on their antioxidant activity. A comprehensive mathematical model was employed to calculate the binding constants, using a fluorescence quenching method, and this was corrected for the inner-filter effect to improve accuracy. We found that a hydroxy group at the 2-position of the benzene ring exerted a positive effect on the affinities, while a 4-hydroxy substituent had a negative influence. Both methylation of the hydroxy groups and replacing the hydroxy groups with methyl groups at the 3- and 4-positions of the benzene ring enhanced the binding affinities. Hydrophobic force and hydrogen bonding were binding forces for the phenolic acids, and their methyl esters, respectively. The antioxidant activity of the HSA-phenolic acid interaction compounds was higher than that of the phenolic acids alone. Copyright © 2017. Published by Elsevier Ltd.

Selective Chemical Labeling of Proteins with Small Fluorescent Molecules Based on Metal-Chelation Methodology

PubMed Central

Soh, Nobuaki

2008-01-01

Site-specific chemical labeling utilizing small fluorescent molecules is a powerful and attractive technique for in vivo and in vitro analysis of cellular proteins, which can circumvent some problems in genetic encoding labeling by large fluorescent proteins. In particular, affinity labeling based on metal-chelation, advantageous due to the high selectivity/simplicity and the small tag-size, is promising, as well as enzymatic covalent labeling, thereby a variety of novel methods have been studied in recent years. This review describes the advances in chemical labeling of proteins, especially highlighting the metal-chelation methodology. PMID:27879749

Fundamentals of affinity cell separations.

PubMed

Zhang, Ye; Lyons, Veronica; Pappas, Dimitri

2018-03-01

Cell separations using affinity methods continue to be an enabling science for a wide variety of applications. In this review, we discuss the fundamental aspects of affinity separation, including the competing forces for cell capture and elution, cell-surface interactions, and models for cell adhesion. Factors affecting separation performance such as bond affinity, contact area, and temperature are presented. We also discuss and demonstrate the effects of nonspecific binding on separation performance. Metrics for evaluating cell separations are presented, along with methods of comparing separation techniques for cell isolation using affinity capture. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

JAK2 JH2 Fluorescence Polarization Assay and Crystal Structures for Complexes with Three Small Molecules.

PubMed

Newton, Ana S; Deiana, Luca; Puleo, David E; Cisneros, José A; Cutrona, Kara J; Schlessinger, Joseph; Jorgensen, William L

2017-06-08

A competitive fluorescence polarization (FP) assay is reported for determining binding affinities of probe molecules with the pseudokinase JAK2 JH2 allosteric site. The syntheses of the fluorescent 5 and 6 used in the assay are reported as well as K d results for 10 compounds, including JNJ7706621, NVP-BSK805, and filgotinib (GLPG0634). X-ray crystal structures of JAK2 JH2 in complex with NVP-BSK805, filgotinib, and diaminopyrimidine 8 elucidate the binding poses.

JAK2 JH2 Fluorescence Polarization Assay and Crystal Structures for Complexes with Three Small Molecules

PubMed Central

2017-01-01

A competitive fluorescence polarization (FP) assay is reported for determining binding affinities of probe molecules with the pseudokinase JAK2 JH2 allosteric site. The syntheses of the fluorescent 5 and 6 used in the assay are reported as well as Kd results for 10 compounds, including JNJ7706621, NVP-BSK805, and filgotinib (GLPG0634). X-ray crystal structures of JAK2 JH2 in complex with NVP-BSK805, filgotinib, and diaminopyrimidine 8 elucidate the binding poses. PMID:28626520

Binding Affinity Effects on Physical Characteristics of a Model Phase-Separated Protein Droplet

NASA Astrophysics Data System (ADS)

Chuang, Sara; Banani, Salman; Rosen, Michael; Brangwynne, Clifford

2015-03-01

Non-membrane bound organelles are associated with a range of biological functions. Several of these structures exhibit liquid-like properties, and may represent droplets of phase-separated RNA and/or proteins. These structures are often enriched in multi-valent molecules, however little is known about the interactions driving the assembly, properties, and function. Here, we address this question using a model multi-valent protein system consisting of repeats of Small Ubiquitin-like Modifier (SUMO) protein and a SUMO-interacting motif (SIM). These proteins undergo phase separation into liquid-like droplets. We combine microrheology and quantitative microscopy to determine affect of binding affinity on the viscosity, density and surface tension of these droplets. We also use fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS) and partitioning experiments to probe the structure and dynamics within these droplets. Our results shed light on how inter-molecular interactions manifests in droplet properties, and lay the groundwork for a comprehensive biophysical picture of intracellular RNA/protein organelles.

Interaction of Chelerythrine with Keyhole Limpet Hemocyanin: a Fluorescence Spectroscopy and Molecular Docking Study

NASA Astrophysics Data System (ADS)

Zhong, M.; Long, R. Q.; Wang, Y. H.; Chen, C. L.

2018-05-01

The quenching mechanism between chelerythrine (CHE) and keyhole limpet hemocyanin (KLH) was investigated using fluorescence spectroscopy and molecular docking. The experiments were conducted at three different temperatures (293, 298, and 303 K). The results revealed that the intrinsic fluorescence of KLH was strongly quenched by CHE through a static quenching mechanism. The thermodynamic parameters (ΔG, ΔH, and ΔS) of the interaction were calculated, indicating that the interaction between CHE and KLH was spontaneous and that van der Waals forces and hydrogen bond formation played major roles in the binding process. The intrinsic fluorescence of the tyrosine and tryptophan residues in KLH was studied by synchronous fluorescence, which suggested that CHE changed the conformation of KLH. Finally, molecular docking was used to obtain detailed information on the binding sites and binding affinities between CHE and KLH.

A new boronic acid fluorescent sensor based on fluorene for monosaccharides at physiological pH

NASA Astrophysics Data System (ADS)

Hosseinzadeh, Rahman; Mohadjerani, Maryam; Pooryousef, Mona; Eslami, Abbas; Emami, Saeed

2015-06-01

Fluorescent boronic acids are very useful fluorescent sensor for detection of biologically important saccharides. Herein we synthesized a new fluorene-based fluorescent boronic acid that shows significant fluorescence changes upon addition of saccharides at physiological pH. Upon addition of fructose, sorbitol, glucose, galactose, ribose, and maltose at different concentration to the solution of 7-(dimethylamino)-9,9-dimethyl-9H-fluoren-2-yl-2-boronic acid (7-DMAFBA, 1), significant decreases in fluorescent intensity were observed. It was found that this boronic acid has high affinity (Ka = 3582.88 M-1) and selectivity for fructose over glucose at pH = 7.4. The sensor 1 showed a linear response toward D-fructose in the concentrations ranging from 2.5 × 10-5 to 4 × 10-4 mol L-1 with the detection limit of 1.3 × 10-5 mol L-1.

Identity, Affinity, Reality: Making the Case for Affinity Groups in Elementary School

ERIC Educational Resources Information Center

Parsons, Julie; Ridley, Kimberly

2012-01-01

Affinity groups are places where students build connections and process "ouch" moments from their classes. Children talk about the isolation they sometimes feel. The relationships students gain through race-based affinity groups enable them to feel less alone with their emotions and help them build a stronger sense of self. At the same…

Fluorescence and confocal imaging of mammalian cells using conjugated oligoelectrolytes with phenylenevinylene core

DOE Office of Scientific and Technical Information (OSTI.GOV)

Milczarek, Justyna; Pawlowska, Roza; Zurawinski, Remigiusz

Over the last few years, considerable efforts are taken, in order to find a molecular fluorescent probe fulfilling their applicability requirements. Due to a good optical properties and affinity to biological structures conjugated oligoelectrolytes (COEs) can be considered as a promising dyes for application in fluorescence-based bioimaging. In this work, we synthetized COEs with phenylenevinylene core (PV-COEs) and applied as fluorescent membranous-specific probes. Cytotoxicity effects of each COE were probed on cancerous and non-cancerous cell types and little to no toxicity effects were observed at the high range of concentrations. The intensity of cell fluorescence following the COE staining wasmore » determined by the photoluminescence analysis and fluorescence activated cell sorting method (FACS). Intercalation of tested COEs into mammalian cell membranes was revealed by fluorescent and confocal microscopy colocalization with commercial dyes specific for cellular structures including mitochondria, Golgi apparatus and endoplasmic reticulum. The phenylenevinylene conjugated oligoelectrolytes have been found to be suitable for fluorescent bioimaging of mammalian cells and membrane-rich organelles. Due to their water solubility coupled with spontaneous intercalation into cells, favorable photophysical features, ease of cell staining, low cytotoxicity and selectivity for membranous structures, PV-COEs can be applied as markers for fluorescence imaging of a variety of cell types.« less

Affinity purification of seminalplasmin and characterization of its interaction with calmodulin.

PubMed Central

Comte, M; Malnoë, A; Cox, J A

1986-01-01

Bull seminalplasmin antagonizes with high potency and selectivity the activating effect of calmodulin on target enzymes [Gietzen & Galla (1985) Biochem. J. 230, 277-280]. In the present paper we establish that seminalplasmin forms a 1:1, Ca2+-dependent and urea-resistant complex with calmodulin. The dissociation constant equals 1.6 nM. In the absence of Ca2+ a low-affinity complex is formed that is disrupted by 4 M-urea. On the basis of these properties, a fast affinity purification of seminalplasmin was developed. The high specificity of seminalplasmin as a calmodulin antagonist was demonstrated for the multipathway-regulated adenylate cyclase of bovine cerebellum. Far-u.v. c.d. properties are consistent with a random form of seminalplasmin in aqueous solution; 23% alpha-helix is induced on interaction with calmodulin. The fluorescence properties of the single tryptophan residue of seminalplasmin are markedly changed on formation of the complex. These studies allowed us to locate tentatively the peptide segment that interacts with calmodulin, and to ascertain the structural homology between seminalplasmin and other calmodulin-binding peptides. Additional material, showing the inhibition of calmodulin-mediated activation of bovine brain phosphodiesterase by melittin and seminalplasmin and also the near-u.v. spectrum of affinity-purified seminalplasmin, has been deposited as supplement SUP 50135 (4 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1986) 233, 5. Images Fig. 2. PMID:3814096

Reaction of Pb(II) and Zn(II) with Ethyl Linoleate To Form Structured Hybrid Inorganic–Organic Complexes: A Model for Degradation in Historic Paint Films

DOE Office of Scientific and Technical Information (OSTI.GOV)

MacDonald, Margaret G.; Palmer, Michael R.; Suchomel, Matthew R.

To investigate soap formation in drying oils in historic paints, the reaction between metal acetates (K +, Zn 2+, Pb 2+) and ethyl linoleate (EL) was studied using optical microscopy, X-ray powder diffraction, and electron microscopy. Pb(II) and Zn(II) react rapidly with EL to form highly structured, spherulitic, luminescent crystallites that aggregate. Evidence from Fourier transform infrared (FTIR) and scanning electron microscopy/energy dispersive X-ray analysis and high-resolution synchrotron powder X-ray diffraction indicates that these are organic-inorganic hybrid complexes or coordination polymers. FTIR absorbance peaks at ca. 1540 cm -1 for Pb(II) and ca. 1580 cm -1 for Zn(II) are consistentmore » with the formation of carboxylate complexes. The complexes formed offer insight into the degradation processes observed in oil paint films, suggesting that soap formation is rapid when metal ions are solubilized and can occur with unsaturated fatty acids that are present in fresh oils. Finally, these complexes may account for the atypical luminescence observed in lead-containing cured oil paint films.« less

Reaction of Pb(II) and Zn(II) with Ethyl Linoleate To Form Structured Hybrid Inorganic–Organic Complexes: A Model for Degradation in Historic Paint Films

DOE Office of Scientific and Technical Information (OSTI.GOV)

MacDonald, Margaret G.; Palmer, Michael R.; Suchomel, Matthew R.

To investigate soap formation in drying oils in historic paints, the reaction between metal acetates (K +, Zn 2+, Pb 2+) and ethyl linoleate (EL) was studied using optical microscopy, X-ray powder diffraction, and electron microscopy. Pb(II) and Zn(II) react rapidly with EL to form highly structured, spherulitic, luminescent crystallites that aggregate. Evidence from Fourier transform infrared (FTIR) and scanning electron microscopy/energy dispersive X-ray analysis and high-resolution synchrotron powder X-ray diffraction indicates that these are organic–inorganic hybrid complexes or coordination polymers. FTIR absorbance peaks at ca. 1540 cm –1 for Pb(II) and ca. 1580 cm –1 for Zn(II) are consistentmore » with the formation of carboxylate complexes. The complexes formed offer insight into the degradation processes observed in oil paint films, suggesting that soap formation is rapid when metal ions are solubilized and can occur with unsaturated fatty acids that are present in fresh oils. These complexes may account for the atypical luminescence observed in lead-containing cured oil paint films.« less

Reaction of Pb(II) and Zn(II) with Ethyl Linoleate To Form Structured Hybrid Inorganic–Organic Complexes: A Model for Degradation in Historic Paint Films

DOE PAGES

MacDonald, Margaret G.; Palmer, Michael R.; Suchomel, Matthew R.; ...

2016-09-07

To investigate soap formation in drying oils in historic paints, the reaction between metal acetates (K +, Zn 2+, Pb 2+) and ethyl linoleate (EL) was studied using optical microscopy, X-ray powder diffraction, and electron microscopy. Pb(II) and Zn(II) react rapidly with EL to form highly structured, spherulitic, luminescent crystallites that aggregate. Evidence from Fourier transform infrared (FTIR) and scanning electron microscopy/energy dispersive X-ray analysis and high-resolution synchrotron powder X-ray diffraction indicates that these are organic-inorganic hybrid complexes or coordination polymers. FTIR absorbance peaks at ca. 1540 cm -1 for Pb(II) and ca. 1580 cm -1 for Zn(II) are consistentmore » with the formation of carboxylate complexes. The complexes formed offer insight into the degradation processes observed in oil paint films, suggesting that soap formation is rapid when metal ions are solubilized and can occur with unsaturated fatty acids that are present in fresh oils. Finally, these complexes may account for the atypical luminescence observed in lead-containing cured oil paint films.« less

Paracetamol and cytarabine binding competition in high affinity binding sites of transporting protein

NASA Astrophysics Data System (ADS)

Sułkowska, A.; Bojko, B.; Równicka, J.; Sułkowski, W. W.

2006-07-01

Paracetamol (acetaminophen, AA) the most popular analgesic drug is commonly used in the treatment of pain in patients suffering from cancer. In our studies, we evaluated the competition in binding with serum albumin between paracetamol (AA) and cytarabine, antyleukemic drug (araC). The presence of one drug can alter the binding affinity of albumin towards the second one. Such interaction can result in changing of the free fraction of the one of these drugs in blood. Two spectroscopic methods were used to determine high affinity binding sites and the competition of the drugs. Basing on the change of the serum albumin fluorescence in the presence of either of the drugs the quenching ( KQ) constants for the araC-BSA and AA-BSA systems were calculated. Analysis of UV difference spectra allowed us to describe the changes in drug-protein complexes (araC-albumin and AA-albumin) induced by the presence of the second drug (AA and araC, respectively). The mechanism of competition between araC and AA has been proposed.

Investigation of the binding affinity in vitamin B12-Bovine serum albumin system using various spectroscopic methods

NASA Astrophysics Data System (ADS)

Makarska-Bialokoz, Magdalena

2017-09-01

The binding affinity between vitamin B12 (VitB12) and bovine serum albumin (BSA) has been investigated in aqueous solution at pH = 7.4, employing UV-vis absorption and steady-state, synchronous and three-dimensional fluorescence spectra techniques. Representative effects noted for BSA intrinsic fluorescence resulting from the interactions with VitB12 confirm the formation of π-π stacked non-covalent and non-fluorescent complexes in the system VitB12-BSA. All the determined parameters, the binding, fluorescence quenching and bimolecular quenching rate constants (of the order of 104 L mol- 1, 103 L mol- 1 and 1011 L mol- 1 s- 1, respectively), as well as Förster resonance energy transfer parameters validate the mechanism of static quenching. The interaction with VitB12 induces folding of the polypeptide chains around Trp residues of BSA, resulting in a more hydrophobic surrounding. Presented outcomes suggest that the addition of VitB12 can lead to the more organized BSA conformation and its more folded tertiary structure, what could influence the physiological functions of bovine serum albumin, notably in case of its overuse or abnormal metabolism.

Selection of specific aptamer against enrofloxacin and fabrication of graphene oxide based label-free fluorescent assay.

PubMed

Dolati, Somayeh; Ramezani, Mohammad; Nabavinia, Maryam Sadat; Soheili, Vahid; Abnous, Khalil; Taghdisi, Seyed Mohammad

2018-05-15

Specific ssDNA aptamers for the antibiotic enrofloxacin (ENR) were isolated from an enriched nucleotide library by SELEX (Systematic Evolution of Ligands by EXponential enrichment) method with high binding affinity. After seven rounds, five aptamers were selected and identified. Apt58 with highest affinity and sensitivity (K d  = 14.19 nM) was employed to develop a label-free fluorescent biosensing approach based on aptamer, graphene oxide (GO) and native fluorescence of ENR for determination of ENR residue in raw milk samples. Under optimized experimental conditions, the linear range was from 5 nM to 250 nM and LOD was calculated to be 3.7 nM, and the recovery rate was between 94.1% and 108.5%. The integration of aptamer and GO in this bioassay provides a promising way for rapid, sensitive and cost-effective detection of ENR in real samples like raw milk. Copyright © 2018 Elsevier Inc. All rights reserved.

Assessment of the binding of hydroxylated polybrominated diphenyl ethers to thyroid hormone transport proteins using a site-specific fluorescence probe.

PubMed

Ren, Xiao M; Guo, Liang-Hong

2012-04-17

Polybrominated diphenyl ethers (PBDEs) have been shown to disrupt thyroid hormone (TH) functions on experimental animals, and one of the proposed disruption mechanisms is the competitive binding of PBDE metabolites to TH transport proteins. In this report, a nonradioactive, site-specific fluorescein-thyroxine (F-T4) conjugate was designed and synthesized as a fluorescence probe to study the binding interaction of hydroxylated PBDEs to thyroxine-binding globulin (TBG) and transthyretin (TTR), two major TH transport proteins in human plasma. Compared with free F-T4, the fluorescence intensity of TTR-bound conjugate was enhanced by as much as 2-fold, and the fluorescence polarization value of TBG-bound conjugate increased by more than 20-fold. These changes provide signal modulation mechanisms for F-T4 as a fluorescence probe. Based on fluorescence quantum yield and lifetime measurements, the fluorescence intensity enhancement was likely due to the elimination of intramolecular fluorescence quenching of fluorescein by T4 after F-T4 was bound to TTR. In circular dichroism and intrinsic tryptophan fluorescence measurements, F-T4 induced similar spectroscopic changes of the proteins as T4 did, suggesting that F-T4 bound to the proteins at the T4 binding site. By using F-T4 as the fluorescence probe in competitive binding assays, 11 OH-PBDEs with different levels of bromination and different hydroxylation positions were assessed for their binding affinity with TBG and TTR, respectively. The results indicate that the binding affinity generally increased with bromine number and OH position also played an important role. 3-OH-BDE-47 and 3'-OH-BDE-154 bound to TTR and TBG even stronger, respectively, than T4. With rising environmental level and high bioaccumulation capability, PBDEs have the potential to disrupt thyroid homeostasis by competitive binding with TH transport proteins.

Simultaneous cryo X-ray ptychographic and fluorescence microscopy of green algae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deng, Junjing; Vine, David J.; Chen, Si

Trace metals play important roles in normal and in disease-causing biological functions. X-ray fluorescence microscopy reveals trace elements with no dependence on binding affinities (unlike with visible light fluorophores) and with improved sensitivity relative to electron probes. However, X-ray fluorescence is not very sensitive for showing the light elements that comprise the majority of cellular material. Here we show that X-ray ptychography can be combined with fluorescence to image both cellular structure and trace element distribution in frozen-hydrated cells at cryogenic temperatures, with high structural and chemical fidelity. Ptychographic reconstruction algorithms deliver phase and absorption contrast images at a resolutionmore » beyond that of the illuminating lens or beam size. Using 5.2-keV X-rays, we have obtained sub-30-nm resolution structural images and similar to 90-nm-resolution fluorescence images of several elements in frozen-hydrated green algae. This combined approach offers a way to study the role of trace elements in their structural context.« less

Tolerance of a Knotted Near-Infrared Fluorescent Protein to Random Circular Permutation.

PubMed

Pandey, Naresh; Kuypers, Brianna E; Nassif, Barbara; Thomas, Emily E; Alnahhas, Razan N; Segatori, Laura; Silberg, Jonathan J

2016-07-12

Bacteriophytochrome photoreceptors (BphP) are knotted proteins that have been developed as near-infrared fluorescent protein (iRFP) reporters of gene expression. To explore how rearrangements in the peptides that interlace into the knot within the BphP photosensory core affect folding, we subjected iRFPs to random circular permutation using an improved transposase mutagenesis strategy and screened for variants that fluoresce. We identified 27 circularly permuted iRFPs that display biliverdin-dependent fluorescence in Escherichia coli. The variants with the brightest whole cell fluorescence initiated translation at residues near the domain linker and knot tails, although fluorescent variants that initiated translation within the PAS and GAF domains were discovered. Circularly permuted iRFPs retained sufficient cofactor affinity to fluoresce in tissue culture without the addition of biliverdin, and one variant displayed enhanced fluorescence when expressed in bacteria and tissue culture. This variant displayed a quantum yield similar to that of iRFPs but exhibited increased resistance to chemical denaturation, suggesting that the observed increase in the magnitude of the signal arose from more efficient protein maturation. These results show how the contact order of a knotted BphP can be altered without disrupting chromophore binding and fluorescence, an important step toward the creation of near-infrared biosensors with expanded chemical sensing functions for in vivo imaging.

Preparation, Characterization, and Antimicrobial Activities of Bimetallic Complexes of Sarcosine with Zn(II) and Sn(IV)

PubMed Central

Arafat, Yasir; Ali, Saqib; Shahzadi, Saira; Shahid, Muhammad

2013-01-01

Heterobimetallic complexes of Zn(II) and Sn(IV) with sarcosine have been synthesized at room temperature under stirring conditions by the reaction of sarcosine and zinc acetate in 2 : 1 molar ratio followed by the stepwise addition of CS2 and organotin(IV) halides, where R = Me, n-Bu, and Ph. The complexes were characterized by elemental analysis, FT-IR and NMR (1H, 13C) spectroscopy. IR data showed that the ligand acts in a bidentate manner. NMR data revealed the four coordinate geometry in solution state. In vitro antimicrobial activities data showed that complexes (3) and (4) were effective against bacterial and fungal strains with few exceptions. PMID:24235910

A mix-and-read drop-based in vitro two-hybrid method for screening high-affinity peptide binders

PubMed Central

Cui, Naiwen; Zhang, Huidan; Schneider, Nils; Tao, Ye; Asahara, Haruichi; Sun, Zhiyi; Cai, Yamei; Koehler, Stephan A.; de Greef, Tom F. A.; Abbaspourrad, Alireza; Weitz, David A.; Chong, Shaorong

2016-01-01

Drop-based microfluidics have recently become a novel tool by providing a stable linkage between phenotype and genotype for high throughput screening. However, use of drop-based microfluidics for screening high-affinity peptide binders has not been demonstrated due to the lack of a sensitive functional assay that can detect single DNA molecules in drops. To address this sensitivity issue, we introduced in vitro two-hybrid system (IVT2H) into microfluidic drops and developed a streamlined mix-and-read drop-IVT2H method to screen a random DNA library. Drop-IVT2H was based on the correlation between the binding affinity of two interacting protein domains and transcriptional activation of a fluorescent reporter. A DNA library encoding potential peptide binders was encapsulated with IVT2H such that single DNA molecules were distributed in individual drops. We validated drop-IVT2H by screening a three-random-residue library derived from a high-affinity MDM2 inhibitor PMI. The current drop-IVT2H platform is ideally suited for affinity screening of small-to-medium-sized libraries (103–106). It can obtain hits within a single day while consuming minimal amounts of reagents. Drop-IVT2H simplifies and accelerates the drop-based microfluidics workflow for screening random DNA libraries, and represents a novel alternative method for protein engineering and in vitro directed protein evolution. PMID:26940078

Facile Synthesis of Molecularly Imprinted Graphene Quantum Dots for the Determination of Dopamine with Affinity-Adjustable.

PubMed

Zhou, Xi; Wang, Anqi; Yu, Chenfei; Wu, Shishan; Shen, Jian

2015-06-10

A facilely prepared fluorescence sensor was developed for dopamine (DA) determination based on polyindole/graphene quantum dots molecularly imprinted polymers (PIn/GQDs@MIPs). The proposed sensor exhibits a high sensitivity with a linear range of 5 × 10(-10) to 1.2 × 10(-6) M and the limit of detection as low as 1 × 10(-10) M in the determination of DA, which is probably due to the tailor-made imprinted cavities for binding DA thought hydrogen bonds between amine groups of DA and oxygen-containing groups of the novel composite. Furthermore, the prepared sensor can rebind DA in dual-type: a low affinity type (noncovalent interaction is off) and a high affinity type (noncovalent interaction is on), and the rebinding interaction can be adjusted by tuning the pH, which shows a unique potential for adjusting the binding interaction while keeping the specificity, allowing for wider applications.

A new boronic acid fluorescent sensor based on fluorene for monosaccharides at physiological pH.

PubMed

Hosseinzadeh, Rahman; Mohadjerani, Maryam; Pooryousef, Mona; Eslami, Abbas; Emami, Saeed

2015-06-05

Fluorescent boronic acids are very useful fluorescent sensor for detection of biologically important saccharides. Herein we synthesized a new fluorene-based fluorescent boronic acid that shows significant fluorescence changes upon addition of saccharides at physiological pH. Upon addition of fructose, sorbitol, glucose, galactose, ribose, and maltose at different concentration to the solution of 7-(dimethylamino)-9,9-dimethyl-9H-fluoren-2-yl-2-boronic acid (7-DMAFBA, 1), significant decreases in fluorescent intensity were observed. It was found that this boronic acid has high affinity (K(a)=3582.88 M(-1)) and selectivity for fructose over glucose at pH=7.4. The sensor 1 showed a linear response toward d-fructose in the concentrations ranging from 2.5×10(-5) to 4×10(-4) mol L(-1) with the detection limit of 1.3×10(-5) mol L(-1). Copyright © 2015 Elsevier B.V. All rights reserved.

Dye-ligand affinity systems.

PubMed

Denizli, A; Pişkin, E

2001-10-30

Dye-ligands have been considered as one of the important alternatives to natural counterparts for specific affinity chromatography. Dye-ligands are able to bind most types of proteins, in some cases in a remarkably specific manner. They are commercially available, inexpensive, and can easily be immobilized, especially on matrices bearing hydroxyl groups. Although dyes are all synthetic in nature, they are still classified as affinity ligands because they interact with the active sites of many proteins mimicking the structure of the substrates, cofactors, or binding agents for those proteins. A number of textile dyes, known as reactive dyes, have been used for protein purification. Most of these reactive dyes consist of a chromophore (either azo dyes, anthraquinone, or phathalocyanine), linked to a reactive group (often a mono- or dichlorotriazine ring). The interaction between the dye ligand and proteins can be by complex combination of electrostatic, hydrophobic, hydrogen bonding. Selection of the supporting matrix is the first important consideration in dye-affinity systems. There are several methods for immobilization of dye molecules onto the support matrix, in which usually several intermediate steps are followed. Both the adsorption and elution steps should carefully be optimized/designed for a successful separation. Dye-affinity systems in the form of spherical sorbents or as affinity membranes have been used in protein separation.

A label-free fluorescent probe for Hg2+ and biothiols based on graphene oxide and Ru-complex

PubMed Central

Wang, Linlin; Yao, Tianming; Shi, Shuo; Cao, Yanlin; Sun, Wenliang

2014-01-01

A novel, selective and sensitive switch-on fluorescent sensor for Hg2+ and switch-off fluorescent probe for biothiols was developed by using [Ru(bpy)2(pip)]2+ as the signal reporter and graphene oxide (GO) as the quencher. Due to the affinity of GO towards single-stranded DNA (ss-DNA) and [Ru(bpy)2(pip)]2+, the three components assembled, resulting in fluorescence quenching. Upon addition of Hg2+, a double-stranded DNA (ds-DNA) via T–Hg2+–T base pairs was formed, and [Ru(bpy)2(pip)]2+ intercalated into the newly formed ds-DNA. Then, [Ru(bpy)2(pip)]2+ and ds-DNA were removed from the surface of GO, resulting in the restoration of fluorescence. Subsequently, upon addition of biothiols, Hg2+ was released from ds-DNA, due to the higher affinity of Hg2+ to the sulfur atoms of biothiols, which could induce ds-DNA unwinding to form ss-DNA. Then ss-DNA and [Ru(bpy)2(pip)]2+ were adsorbed on the surface of GO, the fluorescence of [Ru(bpy)2(pip)]2+ was quenched again. Therefore, the changes in emission intensity of [Ru(bpy)2(pip)]2+ directly correlated to the amount of detection target (Hg2+ or biothiols) in solution. The assay exhibited high sensitivity and selectivity, with the limits of detection for Hg2+, cysteine (Cys) and glutathione (GSH) to be 2.34 nM, 6.20 nM and 4.60 nM, respectively. PMID:24936798

Metal selectivity of the E. coli nickel metallochaperone, SlyD

PubMed Central

Kaluarachchi, Harini; Siebel, Judith F.; Kaluarachchi-Duffy, Supipi; Krecisz, Sandra; Sutherland, Duncan E. K.; Stillman, Martin J.; Zamble, Deborah B.

2012-01-01

SlyD is a Ni(II)-binding protein that contributes to nickel homeostasis in Escherichia coli. The C-terminal domain of SlyD contains a rich variety of metal-binding amino acids, suggesting broader metal-binding capabilities, and previous work demonstrated that the protein can coordinate several types of first row transition metals. However, the binding of SlyD to metals other than Ni(II) has not been previously characterized. To further our understanding of the in vitro metal-binding activity of SlyD and how it correlates with the in vivo function of this protein, the interactions between SlyD and the series of biologically relevant transition metals Mn(II), Fe(II), Co(II), Cu(I) and Zn(II) were examined by using a combination of optical spectroscopy and mass spectrometry. SlyD binding to Mn(II) or to Fe(II) ions was not detected but the protein coordinates multiple ions of Co(II), Zn(II) and Cu(I) with appreciable affinities (KD ≤ nM), highlighting the promiscuous nature of this protein. The order of affinities of SlyD for the metals examined is Mn(II), Fe(II) < Co(II) < Ni(II) ~ Zn(II) ≪ Cu(I). Although the purified protein is unable to overcome the large thermodynamic preference for Cu(I) and exclude Zn(II) chelation in the presence of Ni(II), in vivo studies reveal a Ni(II)-specific function for the protein. Furthermore, these latter experiments support a specific role for SlyD as a [NiFe]-hydrogenase enzyme maturation factor. The implications of the divergence between the metal selectivity of SlyD in vitro and the specific activity in vivo are discussed. PMID:22047179

Affinity-aware checkpoint restart

DOE PAGES

Saini, Ajay; Rezaei, Arash; Mueller, Frank; ...

2014-12-08

Current checkpointing techniques employed to overcome faults for HPC applications result in inferior application performance after restart from a checkpoint for a number of applications. This is due to a lack of page and core affinity awareness of the checkpoint/restart (C/R) mechanism, i.e., application tasks originally pinned to cores may be restarted on different cores, and in case of non-uniform memory architectures (NUMA), quite common today, memory pages associated with tasks on a NUMA node may be associated with a different NUMA node after restart. Here, this work contributes a novel design technique for C/R mechanisms to preserve task-to-core mapsmore » and NUMA node specific page affinities across restarts. Experimental results with BLCR, a C/R mechanism, enhanced with affinity awareness demonstrate significant performance benefits of 37%-73% for the NAS Parallel Benchmark codes and 6-12% for NAMD with negligible overheads instead of up to nearly four times longer an execution times without affinity-aware restarts on 16 cores.« less

Affinity-aware checkpoint restart

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saini, Ajay; Rezaei, Arash; Mueller, Frank

Current checkpointing techniques employed to overcome faults for HPC applications result in inferior application performance after restart from a checkpoint for a number of applications. This is due to a lack of page and core affinity awareness of the checkpoint/restart (C/R) mechanism, i.e., application tasks originally pinned to cores may be restarted on different cores, and in case of non-uniform memory architectures (NUMA), quite common today, memory pages associated with tasks on a NUMA node may be associated with a different NUMA node after restart. Here, this work contributes a novel design technique for C/R mechanisms to preserve task-to-core mapsmore » and NUMA node specific page affinities across restarts. Experimental results with BLCR, a C/R mechanism, enhanced with affinity awareness demonstrate significant performance benefits of 37%-73% for the NAS Parallel Benchmark codes and 6-12% for NAMD with negligible overheads instead of up to nearly four times longer an execution times without affinity-aware restarts on 16 cores.« less

Enhanced fluorescence of tetrasulfonated zinc phthalocyanine by graphene quantum dots and its application in molecular sensing/imaging.

PubMed

Wang, Jian; Zhang, Yanjun; Ye, Jiqing; Jiang, Zhou

2017-06-01

When excited at 435 nm, tetra-sulfonate zinc phthalocyanine (ZnPcS 4 ) emitted dual fluorescence at 495 and 702 nm. The abnormal fluorescence at 495 nm was experimentally studied and analyzed in detail for the first time. The abnormal fluorescence at 495 nm was deduced to originate from triplet-triplet (T-T) energy transfer of excited phthalocyanine ( 3 *ZnPcS 4 ). Furthermore, graphene quantum dots (GQDs) enhanced the 495 nm fluorescence quantum yield (Q) of ZnPcS 4 . The fluorescence properties of ZnPcS 4 -GQDs conjugate were retained in a cellular environment. Based on the fluorescence of ZnPcS 4 -GQDs conjugate, we designed and prepared an Apt29/thrombin/Apt15 sandwich thrombin sensor with high specificity and affinity. This cost-saving, simple operational sensing strategy can be extended to use in sensing/imaging of other biomolecules. Copyright © 2016 John Wiley & Sons, Ltd.

Fluorescence lifetime analysis and effect of magnesium ions on binding of NADH to human aldehyde dehydrogenase 1

USDA-ARS?s Scientific Manuscript database

Aldehyde dehydrogenase 1 (ALDH1) catalyzes oxidation of toxic aldehydes to carboxylic acids. Physiologic levels of Mg2+ ions influence ALDH1 activity in part by increasing NADH binding affinity to the enzyme thus reducing activity. By using time-resolved fluorescence spectroscopy, we have resolved t...

Selective and Sensitive Fluorescent Detection of Picric Acid by New Pyrene and Anthracene Based Copper Complexes.

PubMed

Reddy, Kumbam Lingeshwar; Kumar, Anabathula Manoj; Dhir, Abhimanew; Krishnan, Venkata

2016-11-01

New pyrene and anthracene based copper complexes 4 and 7 respectively were designed, synthesized and characterized. The fluorescence behaviour of both 4 and 7 were evaluated towards nitro aromatics and anions. Both 4 and 7 possess high selectivity for the detection of well-known explosive picric acid (PA) by showing maximum fluorescence affinity. Furthermore, complex 4 showed similar sensing efficiency towards PA at different pH ranges. It was also used for real world applications, as illustrated by the very fast detection of PA from soil samples observed directly by naked eye.

An aptamer-based fluorescence bio-sensor for chiral recognition of arginine enantiomers

NASA Astrophysics Data System (ADS)

Yuan, Haiyan; Huang, Yunmei; Yang, Jidong; Guo, Yuan; Zeng, Xiaoqing; Zhou, Shang; Cheng, Jiawei; Zhang, Yuhui

2018-07-01

In this study, a novel aptamer - based fluorescence bio-sensor (aptamer-AuNps) was developed for chiral recognition of arginine (Arg) enantiomers based on aptamer and gold nanoparticles (AuNps). Carboxyfluorescein (FAM) labeled aptamers (Apt) were absorbed on AuNps and their fluorescence intensity could be significantly quenched by AuNps based on fluorescence resonance energy transfer (FRET). Once D-Arg or L-Arg were added into the above solution, the aptamer specifically bind to Arg enantiomers and released from AuNps, so the fluorescence intensity of D-Arg system and L-Arg system were all enhanced. The affinity of Apt to L-Arg is tighter to D-Arg, so the enhanced fluorescence signals of L-Arg system was stronger than D-Arg system. What's more, the enhanced fluorescence were directly proportional to the concentration of D-Arg and L-Arg ranging from 0-300 nM and 0-400 nM with related coefficients of 0.9939 and 0.9952, respectively. Furthermore, the method was successfully applied to detection L-Arg in human urine samples with satisfactory results. Eventually, a simple "OR" logic gate with D-Arg &L-Arg as inputs and AuNps aggregation state as outputs was fabricated, which can help us understand the chiral recognition process deeply.

SPE coupled to AAS trace determination of Cd(II) and Zn(II) in food samples using amine functionalized GMA-MMA-EGDMA terpolymer: Isotherm and kinetic studies.

PubMed

Islam, Aminul; Kumar, Suneel; Zaidi, Noushi; Ahmad, Hilal

2016-12-15

An ethylenediamine functionalized glycidyl methacrylate (GMA) based terpolymeric chelating resin was synthesized for the separation and preconcentration of Cd(II) and Zn(II) by SPE from bread, rice and fruit juice prior to FAAS determination. The resin was characterized by FT-IR, TGA/DTA, SEM, BET analysis and EDS. Synthesized resin shows a good capacity of 53.96mgg(-1) for Cd(II) and 24.19mgg(-1) for Zn(II) at pH 8.0. Five isotherm equilibrium models were studied to explain the sorption phenomenon out of which Langmuir, Dubinin-Radushkevich, Scatchard and Temkin models were found to be the best fitted. The limit of detection (LOD) and limit of quantification (LOQ) were observed to be 1.5 and 5.1μgL(-1) for Cd and 1.2 and 4.1μgL(-1) for Zn. The reliability of the method was investigated by the analysis of SRM and the recovery of analytes from various spiked food samples. Copyright © 2016 Elsevier Ltd. All rights reserved.

2017 Guralp Affinity Digitizer Evaluation.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Merchant, Bion J.

Sandia National Laboratories has tested and evaluated two Guralp Affinity digitizers. The Affinity digitizers are intended to record sensor output for seismic and infrasound monitoring applications. The purpose of this digitizer evaluation is to measure the performance characteristics in such areas as power consumption, input impedance, sensitivity, full scale, self- noise, dynamic range, system noise, response, passband, and timing. The Affinity digitizers are being evaluated for potential use in the International Monitoring System (IMS) of the Comprehensive Nuclear Test-Ban-Treaty Organization (CTBTO).

Calcium Sensitive Fluorescent Dyes Fluo-4 and Fura Red under Pressure: Behaviour of Fluorescence and Buffer Properties under Hydrostatic Pressures up to 200 MPa

PubMed Central

Vass, H.; Reischl, B.; Allen, R. J.; Friedrich, O.

2016-01-01

The fluorescent Ca2+ sensitive dyes Fura Red (ratiometric) and Fluo-4 (non-ratiometric) are widely utilized for the optical assessment of Ca2+ fluctuations in vitro as well as in situ. The fluorescent behavior of these dyes is strongly depends on temperature, pH, ionic strength and pressure. It is crucial to understand the response of these dyes to pressure when applying calcium imaging technologies in the field of high pressure bioscience. Therefore, we use an optically accessible pressure vessel to pressurize physiological Ca2+-buffered solutions at different fixed concentrations of free Ca2+ (1 nM to 25.6 μM) and a specified dye concentration (12 μM) to pressures of 200 MPa, and record dye fluorescence intensity. Our results show that Fluo-4 fluorescence intensity is reduced by 31% per 100 MPa, the intensity of Fura Red is reduced by 10% per 100 MPa. The mean reaction volume for the dissociation of calcium from the dye molecules Δdv¯ is determined to -17.8 ml mol-1 for Fluo-4 and -21.3 ml mol-1 for Fura Red. Additionally, a model is presented that is used to correct for pressure-dependent changes in pH and binding affinity of Ca2+ to EGTA, as well as to determine the influence of these changes on dye fluorescence. PMID:27764134

Calcium Sensitive Fluorescent Dyes Fluo-4 and Fura Red under Pressure: Behaviour of Fluorescence and Buffer Properties under Hydrostatic Pressures up to 200 MPa.

PubMed

Schneidereit, D; Vass, H; Reischl, B; Allen, R J; Friedrich, O

2016-01-01

The fluorescent Ca2+ sensitive dyes Fura Red (ratiometric) and Fluo-4 (non-ratiometric) are widely utilized for the optical assessment of Ca2+ fluctuations in vitro as well as in situ. The fluorescent behavior of these dyes is strongly depends on temperature, pH, ionic strength and pressure. It is crucial to understand the response of these dyes to pressure when applying calcium imaging technologies in the field of high pressure bioscience. Therefore, we use an optically accessible pressure vessel to pressurize physiological Ca2+-buffered solutions at different fixed concentrations of free Ca2+ (1 nM to 25.6 μM) and a specified dye concentration (12 μM) to pressures of 200 MPa, and record dye fluorescence intensity. Our results show that Fluo-4 fluorescence intensity is reduced by 31% per 100 MPa, the intensity of Fura Red is reduced by 10% per 100 MPa. The mean reaction volume for the dissociation of calcium from the dye molecules [Formula: see text] is determined to -17.8 ml mol-1 for Fluo-4 and -21.3 ml mol-1 for Fura Red. Additionally, a model is presented that is used to correct for pressure-dependent changes in pH and binding affinity of Ca2+ to EGTA, as well as to determine the influence of these changes on dye fluorescence.

G-quadruplex enhanced fluorescence of DNA-silver nanoclusters and their application in bioimaging

NASA Astrophysics Data System (ADS)

Zhu, Jinbo; Zhang, Libing; Teng, Ye; Lou, Baohua; Jia, Xiaofang; Gu, Xiaoxiao; Wang, Erkang

2015-07-01

Guanine proximity based fluorescence enhanced DNA-templated silver nanoclusters (AgNCs) have been reported and applied for bioanalysis. Herein, we studied the G-quadruplex enhanced fluorescence of DNA-AgNCs and gained several significant conclusions, which will be helpful for the design of future probes. Our results demonstrate that a G-quadruplex can also effectively stimulate the fluorescence potential of AgNCs. The major contribution of the G-quadruplex is to provide guanine bases, and its special structure has no measurable impact. The DNA-templated AgNCs were further analysed by native polyacrylamide gel electrophoresis and the guanine proximity enhancement mechanism could be visually verified by this method. Moreover, the fluorescence emission of C3A (CCCA)4 stabilized AgNCs was found to be easily and effectively enhanced by G-quadruplexes, such as T30695, AS1411 and TBA, especially AS1411. Benefiting from the high brightness of AS1411 enhanced DNA-AgNCs and the specific binding affinity of AS1411 for nucleolin, the AS1411 enhanced AgNCs can stain cancer cells for bioimaging.Guanine proximity based fluorescence enhanced DNA-templated silver nanoclusters (AgNCs) have been reported and applied for bioanalysis. Herein, we studied the G-quadruplex enhanced fluorescence of DNA-AgNCs and gained several significant conclusions, which will be helpful for the design of future probes. Our results demonstrate that a G-quadruplex can also effectively stimulate the fluorescence potential of AgNCs. The major contribution of the G-quadruplex is to provide guanine bases, and its special structure has no measurable impact. The DNA-templated AgNCs were further analysed by native polyacrylamide gel electrophoresis and the guanine proximity enhancement mechanism could be visually verified by this method. Moreover, the fluorescence emission of C3A (CCCA)4 stabilized AgNCs was found to be easily and effectively enhanced by G-quadruplexes, such as T30695, AS1411 and TBA, especially

Organization of fluorescent cholesterol analogs in lipid bilayers - lessons from cyclodextrin extraction.

PubMed

Milles, Sigrid; Meyer, Thomas; Scheidt, Holger A; Schwarzer, Roland; Thomas, Lars; Marek, Magdalena; Szente, Lajos; Bittman, Robert; Herrmann, Andreas; Günther Pomorski, Thomas; Huster, Daniel; Müller, Peter

2013-08-01

To characterize the structure and dynamics of cholesterol in membranes, fluorescent analogs of the native molecule have widely been employed. The cholesterol content in membranes is in general manipulated by using water-soluble cyclodextrins. Since the interactions between cyclodextrins and fluorescent-labeled cholesterol have not been investigated in detail so far, we have compared the cyclodextrin-mediated membrane extraction of three different fluorescent cholesterol analogs (one bearing a NBD and two bearing BODIPY moieties). Extraction of these analogs was followed by measuring the Förster resonance energy transfer between a rhodamine moiety linked to phosphatidylethanolamine and the labeled cholesterol. The extraction kinetics revealed that the analogs are differently extracted from membranes. We examined the orientation of the analogs within the membrane and their influence on lipid condensation using NMR and EPR spectroscopies. Our data indicate that the extraction of fluorescent sterols from membranes is determined by several parameters, including their impact on lipid order, their hydrophobicity, their intermolecular interactions with surrounding lipids, their orientation within the bilayer, and their affinity with the exogenous acceptor. Copyright © 2013 Elsevier B.V. All rights reserved.

Evaluation of a metalloporphyrin (THPPMnCl) for necrosis-affinity in rat models of necrosis.

PubMed

Li, Yue; Liu, Xuejiao; Zhang, Dongjian; Lou, Bin; Peng, Fei; Wang, Xiaoning; Shan, Xin; Jiang, Cuihua; Gao, Meng; Sun, Ziping; Ni, Yicheng; Huang, Dejian; Zhang, Jian

2015-12-01

The combination of an (13I)I-labeled necrosis-targeting agent (NTA) with a vascular disrupting agent is a novel and potentially powerful technique for tumor necrosis treatment (TNT). The purpose of this study was to evaluate a NTA candidate, THPPMnCl, using (131)I isotope for tracing its biodistribution and necrosis affinity. (131)I-THPPMnCl was intravenously injected in rat models with liver, muscle, and tumor necrosis and myocardial infarction (MI), followed by investigations with macroscopic autoradiography, triphenyltetrazolium chloride (TTC) histochemical staining, fluorescence microscopy and H&E stained histology for up to 9 days. (131)I-THPPMnCl displayed a long-term affinity for all types of necrosis and accumulation in the mononuclear phagocytic system especially in the liver. Autoradiograms and TTC staining showed a good targetability of (131)I-THPPMnCl for MI. These findings indicate the potential of THPPMnCl for non-invasive imaging assessment of necrosis, such as in MI. However, (13I)I-THPPMnCl is unlikely suitable for TNT due to its long-term retention in normal tissues.

Measuring Norfloxacin Binding to Trypsin Using a Fluorescence Quenching Assay in an Upper-Division, Integrated Laboratory Course

ERIC Educational Resources Information Center

Hicks, Katherine A.

2016-01-01

Fluorescence quenching assays are often used to measure dissociation constants that quantify the binding affinity between small molecules and proteins. In an upper-division undergraduate laboratory course, where students work on projects using a guided inquiry-based approach, a binding titration experiment at physiological pH is performed to…

A new photoactive Ru(II)tris(2,2'-bipyridine) templated Zn(II) benzene-1,4-dicarboxylate metal organic framework: structure and photophysical properties.

PubMed

Whittington, Christi L; Wojtas, Lukasz; Gao, Wen-Yang; Ma, Shengqian; Larsen, Randy W

2015-03-28

It has now been demonstrated that Ru(ii)tris(2,2'-bipyridine) (RuBpy) can be utilized to template the formation of new metal organic framework (MOF) materials containing crystallographically resolved RuBpy clusters with unique photophysical properties. Two such materials, RWLC-1 and RWLC-2, have now been reported from our laboratory and are composed of RuBpy encapsulated in MOFs composed of Zn(ii) ions and 1,3,5-tris(4-carboxyphenyl)benzene ligands (C. L. Whittington, L. Wojtas and R. W. Larsen, Inorg. Chem., 2014, 53, 160-166). Here, a third RuBpy templated photoactive MOF is described (RWLC-3) that is derived from the reaction between Zn(ii) ions and 1,4-dicarboxybenzene in the presence of RuBpy. Single Crystal X-ray diffraction studies determined the position of RuBpy cations within the crystal lattice. The RWLC-3 structure is described as a 2-fold interpenetrated pillared honeycomb network (bnb) containing crystallographically resolved RuBpy clusters. The two bnb networks are weakly interconnected. The encapsulated RuBpy exhibits two emission decay lifetimes (τ-fast = 120 ns, τ-slow = 453 ns) and a bathochromic shift in the steady state emission spectrum relative to RuBpy in ethanol.

A novel expression system for intracellular production and purification of recombinant affinity-tagged proteins in Aspergillus niger.

PubMed

Roth, Andreas H F J; Dersch, Petra

2010-03-01

A set of different integrative expression vectors for the intracellular production of recombinant proteins with or without affinity tag in Aspergillus niger was developed. Target genes can be expressed under the control of the highly efficient, constitutive pkiA promoter or the novel sucrose-inducible promoter of the beta-fructofuranosidase (sucA) gene of A. niger in the presence or absence of alternative carbon sources. All expression plasmids contain an identical multiple cloning sequence that allows parallel construction of N- or C-terminally His6- and StrepII-tagged versions of the target proteins. Production of two heterologous model proteins, the green fluorescence protein and the Thermobifida fusca hydrolase, proved the functionality of the vector system. Efficient production and easy detection of the target proteins as well as their fast purification by a one-step affinity chromatography, using the His6- or StrepII-tag sequence, was demonstrated.

Plasmonically amplified bioassay - Total internal reflection fluorescence vs. epifluorescence geometry.

PubMed

Hageneder, Simone; Bauch, Martin; Dostalek, Jakub

2016-08-15

This paper investigates plasmonic amplification in two commonly used optical configurations for fluorescence readout of bioassays - epifluorescence (EPF) and total internal reflection fluorescence (TIRF). The plasmonic amplification in the EPF configuration was implemented by using crossed gold diffraction grating and Kretschmann geometry of attenuated total reflection method (ATR) was employed in the TIRF configuration. Identical assay, surface architecture for analyte capture, and optics for the excitation, collection and detection of emitted fluorescence light intensity were used in both TIRF and EPF configurations. Simulations predict that the crossed gold diffraction grating (EPF) can amplify the fluorescence signal by a factor of 10(2) by the combination of surface plasmon-enhanced excitation and directional surface plasmon-coupled emission in the red part of spectrum. This factor is about order of magnitude higher than that predicted for the Kretschmann geometry (TIRF) which only took advantage of the surface plasmon-enhanced excitation. When applied for the readout of sandwich interleukin 6 (IL-6) immunoassay, the plasmonically amplified EPF geometry designed for Alexa Fluor 647 labels offered 4-times higher fluorescence signal intensity compared to TIRF. Interestingly, both geometries allowed reaching the same detection limit of 0.4pM despite of the difference in the fluorescence signal enhancement. This is attributed to inherently lower background of fluorescence signal for TIRF geometry compared to that for EPF which compensates for the weaker fluorescence signal enhancement. The analysis of the inflammation biomarker IL-6 in serum at medically relevant concentrations and the utilization of plasmonic amplification for the fluorescence measurement of kinetics of surface affinity reactions are demonstrated for both EPF and TIRF readout. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Antisymmetric tensor generalizations of affine vector fields.

PubMed

Houri, Tsuyoshi; Morisawa, Yoshiyuki; Tomoda, Kentaro

2016-02-01

Tensor generalizations of affine vector fields called symmetric and antisymmetric affine tensor fields are discussed as symmetry of spacetimes. We review the properties of the symmetric ones, which have been studied in earlier works, and investigate the properties of the antisymmetric ones, which are the main theme in this paper. It is shown that antisymmetric affine tensor fields are closely related to one-lower-rank antisymmetric tensor fields which are parallelly transported along geodesics. It is also shown that the number of linear independent rank- p antisymmetric affine tensor fields in n -dimensions is bounded by ( n + 1)!/ p !( n - p )!. We also derive the integrability conditions for antisymmetric affine tensor fields. Using the integrability conditions, we discuss the existence of antisymmetric affine tensor fields on various spacetimes.

Anti-inflammatory drugs interacting with Zn(II), Cd(II) and Pt(II) metal ions.

PubMed

Dendrinou-Samara, C; Tsotsou, G; Ekateriniadou, L V; Kortsaris, A H; Raptopoulou, C P; Terzis, A; Kyriakidis, D A; Kessissoglou, D P

1998-09-01

Complexes of Zn(II), Cd(II) and Pt(II) metal ions with the anti-inflammatory drugs, 1-methyl-5-(p-toluoyl)-1H-pyrrole-2-acetic acid (Tolmetin), alpha-methyl-4-(2-methylpropyl)benzeneacetic acid (Ibuprofen), 6-methoxy-alpha-methylnaphthalene-2-acetic acid (Naproxen) and 1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indole-3-acetic acid (indomethacin) have been synthesized and characterized. In the structurally characterized Cd(naproxen)2 complex the anti-inflammatory drugs acts as bidentate chelate ligand coordinatively bound to metal ions through the deprotonated carboxylate group. Crystal data for 1: [C32H26O8Cd], orthorhombic, space group P22(1)2(1), a = 5.693(2) (A), b = 8.760(3) (A), c = 30.74(1) (A), V = 1533(1) A3, Z = 2. Antibacterial and growth inhibitory activity is higher than that of the parent ligands or the platinum(II) diamine compounds.

The Hexahistidine Motif of Host-Defense Protein Human Calprotectin Contributes to Zinc Withholding and Its Functional Versatility.

PubMed

Nakashige, Toshiki G; Stephan, Jules R; Cunden, Lisa S; Brophy, Megan Brunjes; Wommack, Andrew J; Keegan, Brenna C; Shearer, Jason M; Nolan, Elizabeth M

2016-09-21

Human calprotectin (CP, S100A8/S100A9 oligomer, MRP-8/MRP-14 oligomer) is an abundant host-defense protein that is involved in the metal-withholding innate immune response. CP coordinates a variety of divalent first-row transition metal ions, which is implicated in its antimicrobial function, and its ability to sequester nutrient Zn(II) ions from microbial pathogens has been recognized for over two decades. CP has two distinct transition-metal-binding sites formed at the S100A8/S100A9 dimer interface, including a histidine-rich site composed of S100A8 residues His17 and His27 and S100A9 residues His91 and His95. In this study, we report that CP binds Zn(II) at this site using a hexahistidine motif, completed by His103 and His105 of the S100A9 C-terminal tail and previously identified as the high-affinity Mn(II) and Fe(II) coordination site. Zn(II) binding at this unique site shields the S100A9 C-terminal tail from proteolytic degradation by proteinase K. X-ray absorption spectroscopy and Zn(II) competition titrations support the formation of a Zn(II)-His6 motif. Microbial growth studies indicate that the hexahistidine motif is important for preventing microbial Zn(II) acquisition from CP by the probiotic Lactobacillus plantarum and the opportunistic human pathogen Candida albicans. The Zn(II)-His6 site of CP expands the known biological coordination chemistry of Zn(II) and provides new insight into how the human innate immune system starves microbes of essential metal nutrients.

Development of binding assays for the SH2 domain of Grb7 and Grb2 using fluorescence polarization.

PubMed

Luzy, Jean-Philippe; Chen, Huixiong; Gril, Brunilde; Liu, Wang-Qing; Vidal, Michel; Perdereau, Dominique; Burnol, Anne-Françoise; Garbay, Christiane

2008-02-01

Adaptor proteins Grb7 and Grb2 have been implicated as being 2 potential therapeutic targets in several human cancers, especially those that overexpress ErbB2. These 2 proteins contain both a SH2 domain (Src homology 2) that binds to phosphorylated tyrosine residues contained within ErbB2 and other specific protein targets. Two assays based on enzyme-linked immunosorbent assay and fluorescence polarization methods have been developed and validated to find and rank inhibitors for both proteins binding to the pY(1139). Fluorescence polarization assays allowed the authors to determine quickly and reproducibly affinities of peptides from low nanomolar to high micromolar range and to compare them directly for Grb7 and Grb2. As a result, the assays have identified a known peptidomimetic Grb2 SH2 inhibitor (mAZ-pTyr-(alphaMe)pTyr-Asn-NH(2)) that exhibits the most potent affinity for the Grb7 SH2 domain described to date.

Structure-affinity relationship of the interaction between phenolic acids and their derivatives and β-lactoglobulin and effect on antioxidant activity.

PubMed

Wu, Simin; Zhang, Yunyue; Ren, Fazheng; Qin, Yinghui; Liu, Jiaxin; Liu, Jingwen; Wang, Qingyu; Zhang, Hao

2018-04-15

In this study, 71 phenolic acids and their derivatives were used to investigate the structure-affinity relationship of β-lactoglobulin binding, and the effect of this interaction on antioxidant activity. Based on a fluorescence quenching method, an improved mathematical model was adopted to calculate the binding constants, with a correction for the inner-filter effect. Hydroxylation at the 3-position increased the affinity of the phenolic acids for β-lactoglobulin, while hydroxylation at the 2- or 4-positions had a negative effect. Complete methylation of all hydroxy groups, except at the 3-position, enhanced the binding affinity. Replacing the hydroxy groups with methyl groups at the 2-position also had a positive effect. Hydrogen bonding was one of the binding forces for the interaction. The antioxidant activity of phenolic acid-β-lactoglobulin complexes was higher than that of phenolic acids alone. These findings provide an understanding of the structure-activity relationship of the interaction between β-lactoglobulin and phenolic acids. Copyright © 2017 Elsevier Ltd. All rights reserved.

Synthesis, spectroscopic characterization, first order nonlinear optical properties and DFT calculations of novel Mn(II), Co(II), Ni(II), Cu(II) and Zn(II) complexes with 1,3-diphenyl-4-phenylazo-5-pyrazolone ligand

NASA Astrophysics Data System (ADS)

Abdel-Latif, Samir A.; Mohamed, Adel A.

2018-02-01

Novel Mn(II), Co(II), Ni(II), Cu(II) and Zn(II) metal ions with 1,3-diphenyl-4-phenylazo-5-pyrazolone (L) have been prepared and characterized using different analytical and spectroscopic techniques. 1:1 Complexes of Mn(II), Co(II) and Zn(II) are distorted octahedral whereas Ni(II) complex is square planar and Cu(II) is distorted trigonal bipyramid. 1:2 Complexes of Mn(II), Co(II), Cu(II) and Zn(II) are distorted trigonal bipyramid whereas Ni(II) complex is distorted tetrahedral. All complexes behave as non-ionic in dimethyl formamide (DMF). The electronic structure and nonlinear optical parameters (NLO) of the complexes were investigated theoretically at the B3LYP/GEN level of theory. Molecular stability and bond strengths have been investigated by applying natural bond orbital (NBO) analysis. The geometries of the studied complexes are non-planner. DFT calculations have been also carried out to calculate the global properties; hardness (η), global softness (S) and electronegativity (χ). The calculated small energy gap between HOMO and LUMO energies shows that the charge transfer occurs within the complexes. The total static dipole moment (μtot), the mean polarizability (<α>), the anisotropy of the polarizability (Δα) and the mean first-order hyperpolarizability (<β>) were calculated and compared with urea as a reference material. The complexes show implying optical properties.

Relationship between Ni(II) and Zn(II) Coordination and Nucleotide Binding by the Helicobacter pylori [NiFe]-Hydrogenase and Urease Maturation Factor HypB*

PubMed Central

Sydor, Andrew M.; Lebrette, Hugo; Ariyakumaran, Rishikesh; Cavazza, Christine; Zamble, Deborah B.

2014-01-01

The pathogen Helicobacter pylori requires two nickel-containing enzymes, urease and [NiFe]-hydrogenase, for efficient colonization of the human gastric mucosa. These enzymes possess complex metallocenters that are assembled by teams of proteins in multistep pathways. One essential accessory protein is the GTPase HypB, which is required for Ni(II) delivery to [NiFe]-hydrogenase and participates in urease maturation. Ni(II) or Zn(II) binding to a site embedded in the GTPase domain of HypB modulates the enzymatic activity, suggesting a mechanism of regulation. In this study, biochemical and structural analyses of H. pylori HypB (HpHypB) revealed an intricate link between nucleotide and metal binding. HpHypB nickel coordination, stoichiometry, and affinity were modulated by GTP and GDP, an effect not observed for zinc, and biochemical evidence suggests that His-107 coordination to nickel toggles on and off in a nucleotide-dependent manner. These results are consistent with the crystal structure of HpHypB loaded with Ni(II), GDP, and Pi, which reveals a nickel site distinct from that of zinc-loaded Methanocaldococcus jannaschii HypB as well as subtle changes to the protein structure. Furthermore, Cys-142, a metal ligand from the Switch II GTPase motif, was identified as a key component of the signal transduction between metal binding and the enzymatic activity. Finally, potassium accelerated the enzymatic activity of HpHypB but had no effect on the other biochemical properties of the protein. Altogether, this molecular level information about HpHypB provides insight into its cellular function and illuminates a possible mechanism of metal ion discrimination. PMID:24338018

Relationship between Ni(II) and Zn(II) coordination and nucleotide binding by the Helicobacter pylori [NiFe]-hydrogenase and urease maturation factor HypB.

PubMed

Sydor, Andrew M; Lebrette, Hugo; Ariyakumaran, Rishikesh; Cavazza, Christine; Zamble, Deborah B

2014-02-14

The pathogen Helicobacter pylori requires two nickel-containing enzymes, urease and [NiFe]-hydrogenase, for efficient colonization of the human gastric mucosa. These enzymes possess complex metallocenters that are assembled by teams of proteins in multistep pathways. One essential accessory protein is the GTPase HypB, which is required for Ni(II) delivery to [NiFe]-hydrogenase and participates in urease maturation. Ni(II) or Zn(II) binding to a site embedded in the GTPase domain of HypB modulates the enzymatic activity, suggesting a mechanism of regulation. In this study, biochemical and structural analyses of H. pylori HypB (HpHypB) revealed an intricate link between nucleotide and metal binding. HpHypB nickel coordination, stoichiometry, and affinity were modulated by GTP and GDP, an effect not observed for zinc, and biochemical evidence suggests that His-107 coordination to nickel toggles on and off in a nucleotide-dependent manner. These results are consistent with the crystal structure of HpHypB loaded with Ni(II), GDP, and Pi, which reveals a nickel site distinct from that of zinc-loaded Methanocaldococcus jannaschii HypB as well as subtle changes to the protein structure. Furthermore, Cys-142, a metal ligand from the Switch II GTPase motif, was identified as a key component of the signal transduction between metal binding and the enzymatic activity. Finally, potassium accelerated the enzymatic activity of HpHypB but had no effect on the other biochemical properties of the protein. Altogether, this molecular level information about HpHypB provides insight into its cellular function and illuminates a possible mechanism of metal ion discrimination.

Differential Uptake Mechanisms of Fluorescent Substrates into Stem-Cell-Derived Serotonergic Neurons.

PubMed

Matthaeus, Friederike; Schloss, Patrick; Lau, Thorsten

2015-12-16

The actions of the neurotransmitters serotonin, dopamine, and norepinephrine are partly terminated by diffusion and in part by their uptake into neurons via the selective, high-affinity transporters for serotonin (SERT), dopamine (DAT), and norepinephrine (NET), respectively. There is also growing evidence that all three monoamines are taken up into neurons by low-affinity, high-capacity organic cation transporters (OCT) and the plasma membrane monoamine transporter (PMAT). Pharmacological characterization of these low-affinity recombinant transporter proteins in heterologous expression systems has revealed that they are not antagonized by classical inhibitors of SERT, DAT, or NET but that decynium-22 (D22) antagonizes OCT3 and PMAT, whereas corticosterone and progesterone selectively inhibit OCT3. Here, we show that SERT, PMAT, and OCT3, but not OCT1 and OCT2, are coexpressed in murine stem cell-derived serotonergic neurons. Using selective antagonists, we provide evidence that uptake of the fluorescent substrates FFN511, ASP+, and 5-HT into stem cell-derived serotonergic neurons is mediated differentially by these transporters and also involves an as yet unknown transport mechanism.

Personality Types and Affinity for Computers

DTIC Science & Technology

1991-03-01

differences on personality dimensions between the respondents, and to explore the relationship between these differences and computer affinity. The results...between the respondents, and to explore the relationship between these differences and computer affinity. The results revealed no significant differences...type to this measure of computer affinity. 2 II. LITERATURZ REVIEW The interest of this study was the relationship between a person’s psychological

Smooth affine shear tight frames: digitization and applications

NASA Astrophysics Data System (ADS)

Zhuang, Xiaosheng

2015-08-01

In this paper, we mainly discuss one of the recent developed directional multiscale representation systems: smooth affine shear tight frames. A directional wavelet tight frame is generated by isotropic dilations and translations of directional wavelet generators, while an affine shear tight frame is generated by anisotropic dilations, shears, and translations of shearlet generators. These two tight frames are actually connected in the sense that the affine shear tight frame can be obtained from a directional wavelet tight frame through subsampling. Consequently, an affine shear tight frame indeed has an underlying filter bank from the MRA structure of its associated directional wavelet tight frame. We call such filter banks affine shear filter banks, which can be designed completely in the frequency domain. We discuss the digitization of affine shear filter banks and their implementations: the forward and backward digital affine shear transforms. Redundancy rate and computational complexity of digital affine shear transforms are also investigated in this paper. Numerical experiments and comparisons in image/video processing show the advantages of digital affine shear transforms over many other state-of-art directional multiscale representation systems.

Nicotianamine forms complexes with Zn(II) in vivo.

PubMed

Trampczynska, Aleksandra; Küpper, Hendrik; Meyer-Klaucke, Wolfram; Schmidt, Holger; Clemens, Stephan

2010-01-01

The non-proteinogenic amino acid nicotianamine (NA) is a major player in plant metal homeostasis. It is known to form complexes with different transition metals in vitro. Available evidence associates NA with translocation of Fe, and possibly other micronutrients, to and between different plant cells and tissues. To date, however, it is still extremely challenging to detect metal-ligand complexes in vivo because tissue disruption immediately changes the chemical environment and thereby the availability of binding partners. In order to overcome this limitation we used various Schizosaccharomyces pombe strains expressing a plant NAS gene to study formation of metal-NA complexes in vivo. Tolerance, accumulation and competition data clearly indicated formation of Zn(ii)-NA but not of Cu(ii)-NA complexes. Zn(ii)-NA was then identified by X-ray absorption spectroscopy (XAS). About half of the cellular Zn was found to be bound by NA in NAS-expressing cells while no NA-like ligands were detected by XAS in control cells not expressing NAS. Given the high conservation of eukaryotic metal homeostasis components, these results strongly suggest the possible existence of Zn(ii)-NA complexes also in planta. Reported observations implicating NA in plant Zn homeostasis would then indeed be attributable to direct interaction of Zn(ii) with NA rather than only indirectly to perturbations in Fe metabolism. Re-evaluation of extended X-ray absorption fine structure (EXAFS) spectra for the Zn hyperaccumulator Thlaspi caerulescens showed that NA is as expected not a major storage ligand for Zn. Instead it is hypothesized to be involved in efficient translocation of Zn to above-ground tissues in hyperaccumulators.

Noninvasive imaging of multiple myeloma using near infrared fluorescent molecular probe

NASA Astrophysics Data System (ADS)

Hathi, Deep; Zhou, Haiying; Bollerman-Nowlis, Alex; Shokeen, Monica; Akers, Walter J.

2016-03-01

Multiple myeloma is a plasma cell malignancy characterized by monoclonal gammopathy and osteolytic bone lesions. Multiple myeloma is most commonly diagnosed in late disease stages, presenting with pathologic fracture. Early diagnosis and monitoring of disease status may improve quality of life and long-term survival for multiple myeloma patients from what is now a devastating and fatal disease. We have developed a near-infrared targeted fluorescent molecular probe with high affinity to the α4β1 integrin receptor (VLA-4)overexpressed by a majority of multiple myeloma cells as a non-radioactive analog to PET/CT tracer currently being developed for human diagnostics. A near-infrared dye that emits about 700 nm was conjugated to a high affinity peptidomimmetic. Binding affinity and specificity for multiple myeloma cells was investigated in vitro by tissue staining and flow cytometry. After demonstration of sensitivity and specificity, preclinical optical imaging studies were performed to evaluate tumor specificity in murine subcutaneous and metastatic multiple myeloma models. The VLA-4-targeted molecular probe showed high affinity for subcutaneous MM tumor xenografts. Importantly, tumor cells specific accumulation in the bone marrow of metastatic multiple myeloma correlated with GFP signal from transfected cells. Ex vivo flow cytometry of tumor tissue and bone marrow further corroborated in vivo imaging data, demonstrating the specificity of the novel agent and potential for quantitative imaging of multiple myeloma burden in these models.

Novel Benzothiazole Derivatives as Fluorescent Probes for Detection of β-Amyloid and α-Synuclein Aggregates.

PubMed

Watanabe, Hiroyuki; Ono, Masahiro; Ariyoshi, Taisuke; Katayanagi, Rikako; Saji, Hideo

2017-08-16

Deposits of β-amyloid (Aβ) and α-synuclein (α-syn) are the hallmark of Alzheimer's disease (AD) and Parkinson's disease (PD), respectively. The detection of these protein aggregates with fluorescent probes is particularly of interest for preclinical studies using fluorescence microscopy on human brain tissue. In this study, we newly designed and synthesized three push-pull benzothiazole (PP-BTA) derivatives as fluorescent probes for detection of Aβ and α-syn aggregates. Fluorescence intensity of all PP-BTA derivatives significantly increased upon binding to Aβ(1-42) and α-syn aggregates in solution. In in vitro saturation binding assays, PP-BTA derivatives demonstrated affinity for both Aβ(1-42) (K d = 40-148 nM) and α-syn (K d = 48-353 nM) aggregates. In particular, PP-BTA-4 clearly stained senile plaques composed of Aβ aggregates in the AD brain section. Moreover, it also labeled Lewy bodies composed of α-syn aggregates in the PD brain section. These results suggest that PP-BTA-4 may serve as a promising fluorescent probe for the detection of Aβ and α-syn aggregates.

Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin

PubMed Central

Nilvebrant, Johan; Åstrand, Mikael; Georgieva-Kotseva, Maria; Björnmalm, Mattias; Löfblom, John; Hober, Sophia

2014-01-01

The epidermal growth factor receptor 2, ERBB2, is a well-validated target for cancer diagnostics and therapy. Recent studies suggest that the over-expression of this receptor in various cancers might also be exploited for antibody-based payload delivery, e.g. antibody drug conjugates. In such strategies, the full-length antibody format is probably not required for therapeutic effect and smaller tumor-specific affinity proteins might be an alternative. However, small proteins and peptides generally suffer from fast excretion through the kidneys, and thereby require frequent administration in order to maintain a therapeutic concentration. In an attempt aimed at combining ERBB2-targeting with antibody-like pharmacokinetic properties in a small protein format, we have engineered bispecific ERBB2-binding proteins that are based on a small albumin-binding domain. Phage display selection against ERBB2 was used for identification of a lead candidate, followed by affinity maturation using second-generation libraries. Cell surface display and flow-cytometric sorting allowed stringent selection of top candidates from pools pre-enriched by phage display. Several affinity-matured molecules were shown to bind human ERBB2 with sub-nanomolar affinity while retaining the interaction with human serum albumin. Moreover, parallel selections against ERBB2 in the presence of human serum albumin identified several amino acid substitutions that dramatically modulate the albumin affinity, which could provide a convenient means to control the pharmacokinetics. The new affinity proteins competed for ERBB2-binding with the monoclonal antibody trastuzumab and recognized the native receptor on a human cancer cell line. Hence, high affinity tumor targeting and tunable albumin binding were combined in one small adaptable protein. PMID:25089830

A fully-integrated aptamer-based affinity assay platform for monitoring astronaut health in space.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Xianbin; Durland, Ross H.; Hecht, Ariel H.

2010-07-01

Here we demonstrate the suitability of robust nucleic acid affinity reagents in an integrated point-of-care diagnostic platform for monitoring proteomic biomarkers indicative of astronaut health in spaceflight applications. A model thioaptamer targeting nuclear factor-kappa B (NF-{kappa}B) is evaluated in an on-chip electrophoretic gel-shift assay for human serum. Key steps of (i) mixing sample with the aptamer, (ii) buffer exchange, and (iii) preconcentration of sample were successfully integrated upstream of fluorescence-based detection. Challenges due to (i) nonspecific interactions with serum, and (ii) preconcentration at a nanoporous membrane are discussed and successfully resolved to yield a robust, rapid, and fully-integrated diagnostic system.

An aptamer-based fluorescence bio-sensor for chiral recognition of arginine enantiomers.

PubMed

Yuan, Haiyan; Huang, Yunmei; Yang, Jidong; Guo, Yuan; Zeng, Xiaoqing; Zhou, Shang; Cheng, Jiawei; Zhang, Yuhui

2018-07-05

In this study, a novel aptamer - based fluorescence bio-sensor (aptamer-AuNps) was developed for chiral recognition of arginine (Arg) enantiomers based on aptamer and gold nanoparticles (AuNps). Carboxyfluorescein (FAM) labeled aptamers (Apt) were absorbed on AuNps and their fluorescence intensity could be significantly quenched by AuNps based on fluorescence resonance energy transfer (FRET). Once d-Arg or l-Arg were added into the above solution, the aptamer specifically bind to Arg enantiomers and released from AuNps, so the fluorescence intensity of d-Arg system and l-Arg system were all enhanced. The affinity of Apt to l-Arg is tighter to d-Arg, so the enhanced fluorescence signals of l-Arg system was stronger than d-Arg system. What's more, the enhanced fluorescence were directly proportional to the concentration of d-Arg and l-Arg ranging from 0-300 nM and 0-400 nM with related coefficients of 0.9939 and 0.9952, respectively. Furthermore, the method was successfully applied to detection l-Arg in human urine samples with satisfactory results. Eventually, a simple "OR" logic gate with d-Arg &l-Arg as inputs and AuNps aggregation state as outputs was fabricated, which can help us understand the chiral recognition process deeply. Copyright © 2018 Elsevier B.V. All rights reserved.

Application of volcanic ash particles for protein affinity purification with a minimized silica-binding tag.

PubMed

Abdelhamid, Mohamed A A; Ikeda, Takeshi; Motomura, Kei; Tanaka, Tatsuya; Ishida, Takenori; Hirota, Ryuichi; Kuroda, Akio

2016-11-01

We recently reported that the spore coat protein, CotB1 (171 amino acids), from Bacillus cereus mediates silica biomineralization and that the polycationic C-terminal sequence of CotB1 (14 amino acids), designated CotB1p, serves as a silica-binding tag when fused to other proteins. Here, we reduced the length of this silica-binding tag to only seven amino acids (SB7 tag: RQSSRGR) while retaining its affinity for silica. Alanine scanning mutagenesis indicated that the three arginine residues in the SB7 tag play important roles in binding to a silica surface. Monomeric l-arginine, at concentrations of 0.3-0.5 M, was found to serve as a competitive eluent to release bound SB7-tagged proteins from silica surfaces. To develop a low-cost, silica-based affinity purification procedure, we used natural volcanic ash particles with a silica content of ∼70%, rather than pure synthetic silica particles, as an adsorbent for SB7-tagged proteins. Using green fluorescent protein, mCherry, and mKate2 as model proteins, our purification method achieved 75-90% recovery with ∼90% purity. These values are comparable to or even higher than that of the commonly used His-tag affinity purification. In addition to low cost, another advantage of our method is the use of l-arginine as the eluent because its protein-stabilizing effect would help minimize alteration of the intrinsic properties of the purified proteins. Our approach paves the way for the use of naturally occurring materials as adsorbents for simple, low-cost affinity purification. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Impaired binding affinity of electronegative low-density lipoprotein (LDL) to the LDL receptor is related to nonesterified fatty acids and lysophosphatidylcholine content.

PubMed

Benítez, Sonia; Villegas, Virtudes; Bancells, Cristina; Jorba, Oscar; González-Sastre, Francesc; Ordóñez-Llanos, Jordi; Sánchez-Quesada, José Luis

2004-12-21

The binding characteristics of electropositive [LDL(+)] and electronegative LDL [LDL(-)] subfractions to the LDL receptor (LDLr) were studied. Saturation kinetic studies in cultured human fibroblasts demonstrated that LDL(-) from normolipemic (NL) and familial hypercholesterolemic (FH) subjects had lower binding affinity than their respective LDL(+) fractions (P < 0.05), as indicated by higher dissociation constant (K(D)) values. FH-LDL(+) also showed lower binding affinity (P < 0.05) than NL-LDL(+) (K(D), sorted from lower to higher affinity: NL-LDL(-), 33.0 +/- 24.4 nM; FH-LDL(-), 24.4 +/- 7.1 nM; FH-LDL(+), 16.6 +/- 7.0 nM; NL-LDL(+), 10.9 +/- 5.7 nM). These results were confirmed by binding displacement studies. The impaired affinity binding of LDL(-) could be attributed to altered secondary and tertiary structure of apolipoprotein B, but circular dichroism (CD) and tryptophan fluorescence (TrpF) studies revealed no structural differences between LDL(+) and LDL(-). To ascertain the role of increased nonesterified fatty acids (NEFA) and lysophosphatidylcholine (LPC) content in LDL(-), LDL(+) was enriched in NEFA or hydrolyzed with secretory phospholipase A(2). Modification of LDL gradually decreased the affinity to LDLr in parallel to the increasing content of NEFA and/or LPC. Modified LDLs with a NEFA content similar to that of LDL(-) displayed similar affinity. ApoB structure studies of modified LDLs by CD and TrpF showed no difference compared to LDL(+) or LDL(-). Our results indicate that NEFA loading or phospholipase A(2) lipolysis of LDL leads to changes that affect the affinity of LDL to LDLr with no major effect on apoB structure. Impaired affinity to the LDLr shown by LDL(-) is related to NEFA and/or LPC content rather than to structural differences in apolipoprotein B.

Characterization of ligand binding to melanocortin 4 receptors using fluorescent peptides with improved kinetic properties.

PubMed

Link, Reet; Veiksina, Santa; Rinken, Ago; Kopanchuk, Sergei

2017-03-15

Melanocortin 4 (MC 4 ) receptors are important drug targets as they regulate energy homeostasis, eating behaviour and sexual functions. The ligand binding process to these G protein-coupled receptors is subject to considerable complexity. Different steps in the complex dynamic regulation can be characterized by ligand binding kinetics. Optimization of these kinetic parameters in terms of on-rate and residence time can increase the rapid onset of drug action and reduce off-target effects. Fluorescence anisotropy (FA) is one of the homogeneous fluorescence-based assays that enable continuous online monitoring of ligand binding kinetics. FA has been implemented for the kinetic study of melanocortin MC 4 receptors expressed on budded baculoviruses. However, the slow dissociation of the fluorescently labelled peptide NDP-α-MSH does not enable reaching equilibrium nor enable more in-depth study of the binding mechanisms. To overcome this problem, two novel red-shifted fluorescent ligands were designed. These cyclized heptapeptide derivatives (UTBC101 and UTBC102) exhibited nanomolar affinity toward melanocortin MC 4 receptors but had relatively different kinetic properties. The dissociation half-lives of UTBC101 (τ 1/2 =160min) and UTBC102 (τ 1/2 =7min) were shorter compared to that what was previously reported for Cy3B-NDP-α-MSH (τ 1/2 =224min). The significantly shorter dissociation half-life of UTBC102 enables equilibrium in screening assays, whereas the higher affinity of UTBC101 helps to resolve a wider range of competitor potencies. These two ligands are suitable for further kinetic screening of novel melanocortin MC 4 receptor specific ligands and could complement each other in these studies. Copyright © 2017 Elsevier B.V. All rights reserved.

A supramolecular Tröger's base derived coordination zinc polymer for fluorescent sensing of phenolic-nitroaromatic explosives in water.

PubMed

Shanmugaraju, Sankarasekaran; Dabadie, Charlyne; Byrne, Kevin; Savyasachi, Aramballi J; Umadevi, Deivasigamani; Schmitt, Wolfgang; Kitchen, Jonathan A; Gunnlaugsson, Thorfinnur

2017-02-01

A V-Shaped 4-amino-1,8-napthalimide derived tetracarboxylic acid linker ( L ; bis-[ N -(1,3-benzenedicarboxylic acid)]-9,18-methano-1,8-naphthalimide-[ b , f ][1,5]diazocine) comprising the Tröger's base (TB) structural motif was rationally designed and synthesised to access a nitrogen-rich fluorescent supramolecular coordination polymer. By adopting the straight forward precipitation method, a new luminescent nanoscale Zn(ii) coordination polymer ( TB-Zn-CP ) was synthesized in quantitative yield using Zn(OAc) 2 ·2H 2 O and tetraacid linker L (1 : 0.5) in DMF at room temperature. The phase-purity of as-synthesised TB-Zn-CP was confirmed by X-ray powder diffraction analysis, infra-red spectroscopy, and elemental analysis. Thermogravimetric analysis suggests that TB-Zn-CP is thermally stable up to 330 °C and the morphological features of TB-Zn-CP was analysed by SEM and AFM techniques. The N 2 adsorption isotherm of thermally activated TB-Zn-CP at 77 K revealed a type-II reversible adsorption isotherm and the calculated Brunauer-Emmett-Teller (BET) surface area was found to be 72 m 2 g -1 . Furthermore, TB-Zn-CP displayed an excellent CO 2 uptake capacity of 76 mg g -1 at 273 K and good adsorption selectivity for CO 2 over N 2 and H 2 . The aqueous suspension of as-synthesized TB-Zn-CP showed strong green fluorescence ( λ max = 520 nm) characteristics due to the internal-charge transfer (ICT) transition and was used as a fluorescent sensor for the discriminative sensing of nitroaromatic explosives. The aqueous suspension of TB-Zn-CP showed the largest quenching responses with high selectivity for phenolic-nitroaromatics (4-NP, 2,4-DNP and PA) even in the concurrent presence of other potentially competing nitroaromatic analytes. The fluorescence titration studies also provide evidence that TB-Zn-CP detects picric acid as low as the parts per billion (26.3 ppb) range. Furthermore, the observed fluorescence quenching responses of TB-Zn-CP towards picric acid

Single-step affinity purification for fungal proteomics.

PubMed

Liu, Hui-Lin; Osmani, Aysha H; Ukil, Leena; Son, Sunghun; Markossian, Sarine; Shen, Kuo-Fang; Govindaraghavan, Meera; Varadaraj, Archana; Hashmi, Shahr B; De Souza, Colin P; Osmani, Stephen A

2010-05-01

A single-step protein affinity purification protocol using Aspergillus nidulans is described. Detailed protocols for cell breakage, affinity purification, and depending on the application, methods for protein release from affinity beads are provided. Examples defining the utility of the approaches, which should be widely applicable, are included.

Ratiometric and turn-on monitoring for heavy and transition metal ions in aqueous solution with a fluorescent peptide sensor.

PubMed

Joshi, Bishnu Prasad; Park, Junwon; Lee, Wan In; Lee, Keun-Hyeung

2009-05-15

A novel fluorescent peptide sensor containing tryptophan (donor) and dansyl fluorophore (acceptor) was synthesized for monitoring heavy and transition metal (HTM) ions on the basis of metal ion binding motif (Cys-X-X-X-Cys). The peptide probe successfully exhibited a turn on and ratiometric response for several heavy metal ions such as Hg(2+), Cd(2+), Pb(2+), Zn(2+), and Ag(+) in aqueous solution. The enhancements of emission intensity were achieved in the presence of the HTM ions by fluorescent resonance energy transfer (FRET) and chelation enhanced fluorescence (CHEF) effects. The detection limits of the sensor for Cd(2+), Pb(2+), Zn(2+), and Ag(+) were lower than the EPA's drinking water maximum contaminant levels (MCL). We described the fluorescent enhancement, binding affinity, and detection limit of the peptide probe for HTM ions.

Colorful protein-based fluorescent probes for collagen imaging.

PubMed

Aper, Stijn J A; van Spreeuwel, Ariane C C; van Turnhout, Mark C; van der Linden, Ardjan J; Pieters, Pascal A; van der Zon, Nick L L; de la Rambelje, Sander L; Bouten, Carlijn V C; Merkx, Maarten

2014-01-01

Real-time visualization of collagen is important in studies on tissue formation and remodeling in the research fields of developmental biology and tissue engineering. Our group has previously reported on a fluorescent probe for the specific imaging of collagen in live tissue in situ, consisting of the native collagen binding protein CNA35 labeled with fluorescent dye Oregon Green 488 (CNA35-OG488). The CNA35-OG488 probe has become widely used for collagen imaging. To allow for the use of CNA35-based probes in a broader range of applications, we here present a toolbox of six genetically-encoded collagen probes which are fusions of CNA35 to fluorescent proteins that span the visible spectrum: mTurquoise2, EGFP, mAmetrine, LSSmOrange, tdTomato and mCherry. While CNA35-OG488 requires a chemical conjugation step for labeling with the fluorescent dye, these protein-based probes can be easily produced in high yields by expression in E. coli and purified in one step using Ni2+-affinity chromatography. The probes all bind specifically to collagen, both in vitro and in porcine pericardial tissue. Some first applications of the probes are shown in multicolor imaging of engineered tissue and two-photon imaging of collagen in human skin. The fully-genetic encoding of the new probes makes them easily accessible to all scientists interested in collagen formation and remodeling.

Mobile Technology Affinity in Renal Transplant Recipients.

PubMed

Reber, S; Scheel, J; Stoessel, L; Schieber, K; Jank, S; Lüker, C; Vitinius, F; Grundmann, F; Eckardt, K-U; Prokosch, H-U; Erim, Y

Medication nonadherence is a common problem in renal transplant recipients (RTRs). Mobile health approaches to improve medication adherence are a current trend, and several medication adherence apps are available. However, it is unknown whether RTRs use these technologies and to what extent. In the present study, the mobile technology affinity of RTRs was analyzed. We hypothesized significant age differences in mobile technology affinity and that mobile technology affinity is associated with better cognitive functioning as well as higher educational level. A total of 109 RTRs (63% male) participated in the cross-sectional study, with an overall mean age of 51.8 ± 14.2 years. The study included the Technology Experience Questionnaire (TEQ) for the assessment of mobile technology affinity, a cognitive test battery, and sociodemographic data. Overall, 57.4% of the patients used a smartphone or tablet and almost 45% used apps. The TEQ sum score was 20.9 in a possible range from 6 (no affinity to technology) to 30 (very high affinity). Younger patients had significantly higher scores in mobile technology affinity. The only significant gender difference was found in having fun with using electronic devices: Men enjoyed technology more than women did. Mobile technology affinity was positively associated with cognitive functioning and educational level. Young adult patients might profit most from mobile health approaches. Furthermore, high educational level and normal cognitive functioning promote mobile technology affinity. This should be kept in mind when designing mobile technology health (mHealth) interventions for RTRs. For beneficial mHealth interventions, further research on potential barriers and desired technologic features is necessary to adapt apps to patients' needs. Copyright © 2017 Elsevier Inc. All rights reserved.

Specific capture and detection of Staphylococcus aureus with high-affinity modified aptamers to cell surface components

PubMed Central

Baumstummler, A; Lehmann, D; Janjic, N; Ochsner, UA

2014-01-01

Slow off-rate modified aptamer (SOMAmer) reagents were generated to several Staphylococcus aureus cell surface-associated proteins via SELEX with multiple modified DNA libraries using purified recombinant or native proteins. High-affinity binding agents with sub-nanomolar Kd's were obtained for staphylococcal protein A (SpA), clumping factors (ClfA, ClfB), fibronectin-binding proteins (FnbA, FnbB) and iron-regulated surface determinants (Isd). Further screening revealed several SOMAmers that specifically bound to Staph. aureus cells from all strains that were tested, but not to other staphylococci or other bacteria. SpA and ClfA SOMAmers proved useful for the selective capture and enrichment of Staph. aureus cells, as shown by culture and PCR, leading to improved limits of detection and efficient removal of PCR inhibitors. Detection of Staph. aureus cells was enhanced by several orders of magnitude when the bacterial cell surface was coated with SOMAmers followed by qPCR of the SOMAmers. Furthermore, fluorescence-labelled SpA SOMAmers demonstrated their utility as direct detection agents in flow cytometry. Significance and Impact of the Study Monitoring for microbial contamination of food, water, nonsterile products or the environment is typically based on culture, PCR or antibodies. Aptamers that bind with high specificity and affinity to well-conserved cell surface epitopes represent a promising novel type of reagents to detect bacterial cells without the need for culture or cell lysis, including for the capture and enrichment of bacteria present at low cell densities and for the direct detection via qPCR or fluorescent staining. PMID:24935714

Facile synthesis, cytotoxicity and bioimaging of Fe(3+) selective fluorescent chemosensor.

PubMed

Saleem, Muhammad; Abdullah, Razack; Ali, Anser; Park, Bong Joo; Choi, Eun Ha; Hong, In Seok; Lee, Ki Hwan

2014-04-01

The designing and development of fluorescent chemosensors have recently been intensively explored for sensitive and specific detection of environmentally and biologically relevant metal ions in aqueous solution and living cells. Herein, we report the photophysical results of alanine substituted rhodamine B derivative 3 having specific binding affinity toward Fe(3+) with micro molar concentration level. Through fluorescence titration at 599nm, we were confirmed that ligand 3 exhibited ratiometric fluorescence response with remarkable enhancement in emission intensity by complexation between 3 and Fe(3+) while it appeared no emission in case of the competitive ions (Sc(3+), Yb(3+), In(3+), Ce(3+), Sm(3+), Cr(3+), Sn(2+), Pb(2+), Ni(2+), Co(2+), Cu(2+), Ba(2+), Ca(2+), Mg(2+), Ag(+), Cs(+), Cu(+), K(+)) in aqueous/methanol (60:40, v/v) at neutral pH. However, the fluorescence as well as colorimetric response of ligand-iron complex solution was quenched by addition of KCN which snatches the Fe(3+) from complex and turn off the sensor confirming the recognition process was reversible. Furthermore, bioimaging studies against L-929 cells (mouse fibroblast cells) and BHK-21 (hamster kidney fibroblast), through confocal fluorescence microscopic experiment indicated that ligand showed good permeability and minimum toxicity against the tested cell lines. Copyright © 2014 Elsevier Ltd. All rights reserved.

Development of tumor-targeted near infrared probes for fluorescence guided surgery.

PubMed

Kelderhouse, Lindsay E; Chelvam, Venkatesh; Wayua, Charity; Mahalingam, Sakkarapalayam; Poh, Scott; Kularatne, Sumith A; Low, Philip S

2013-06-19

Complete surgical resection of malignant disease is the only reliable method to cure cancer. Unfortunately, quantitative tumor resection is often limited by a surgeon's ability to locate all malignant disease and distinguish it from healthy tissue. Fluorescence-guided surgery has emerged as a tool to aid surgeons in the identification and removal of malignant lesions. While nontargeted fluorescent dyes have been shown to passively accumulate in some tumors, the resulting tumor-to-background ratios are often poor, and the boundaries between malignant and healthy tissues can be difficult to define. To circumvent these problems, our laboratory has developed high affinity tumor targeting ligands that bind to receptors that are overexpressed on cancer cells and deliver attached molecules selectively into these cells. In this study, we explore the use of two tumor-specific targeting ligands (i.e., folic acid that targets the folate receptor (FR) and DUPA that targets prostate specific membrane antigen (PSMA)) to deliver near-infrared (NIR) fluorescent dyes specifically to FR and PSMA expressing cancers, thereby rendering only the malignant cells highly fluorescent. We report here that all FR- and PSMA-targeted NIR probes examined bind cultured cancer cells in the low nanomolar range. Moreover, upon intravenous injection into tumor-bearing mice with metastatic disease, these same ligand-NIR dye conjugates render receptor-expressing tumor tissues fluorescent, enabling their facile resection with minimal contamination from healthy tissues.

Interaction with biomacromolecules and antiproliferative activities of Mn(II), Ni(II), Zn(II) complexes of demethylcantharate and 2,2'-bipyridine

NASA Astrophysics Data System (ADS)

Zhang, Fan; Lin, Qiu-Yue; Hu, Wan-Li; Song, Wen-Ji; Shen, Shu-Ting; Gui, Pan

2013-06-01

Three new transition metal complexes [Mn2(DCA)2(bipy)2]·5H2O (1), [M2(DCA)2(bipy)2(H2O)]·10H2O(M = Ni(II)(2);Zn(II)(3)), (DCA = demethylcantharate, 7-oxabicyclo[2,2,1]heptane-2,3-dicarboxylate, C8H8O5) were synthesized and characterized by elemental analysis, molar conductance, infrared spectra and X-ray diffraction techniques. Each metal ion was six-coordinated in complexes. Complex 1 has a Mn2O2 center. Complexes 2 and 3 have asymmetric binuclear structure. Great amount of intermolecular hydrogen-bonding and π-π* stacking interactions were formed in these complex structures. The DNA-binding properties of complexes were investigated by electronic absorption spectra and viscosity measurements. The DNA binding constants Kb/(L mol-1) were 1.71 × 104 (1), 2.62 × 104 (2) and 1.59 × 104 (3) at 298 K. The complexes could quench the intrinsic fluorescence of bovine serum albumin (BSA) strongly through static quenching. The protein binding constants Ka/(L mol-1) were 7.27 × 104 (1), 4.55 × 104 (2) and 7.87 × 104 L mol-1 (3) and binding site was one. The complexes bind more tightly with DNA and BSA than with ligands. Complexes 1 and 3 had stronger inhibition ratios than Na2(DCA) against human hepatoma cells (SMMC-7721) lines and human gastric cancer cells (MGC80-3) lines in vitro. Complex 3 showed the strongest antiproliferative activity against SMMC-7721 (IC50 = 29.46 ± 2.12 μmol L-1) and MGC80-3 (IC50 = 27.02 ± 2.38 μmol L-1), which shows potential in anti-cancer drug development.

One Dimensional Coordination Polymer of Zn(II) for Developing Multifunctional Nanoparticles.

PubMed

Agarwal, Rashmi A

2017-10-16

A variety of nanoparticles (NPs) including Ag, Au, Pd, Cr and mixed Cu/Fe have been synthesized in a non-activated (without solvent removal) one dimensional coordination polymer (CP) of Zn(II) via two different mechanisms, acid formation and redox activity of the framework. Main driving force to grow these NPs within the cavities of CP is the presence of free oxygens of one of the monodentate carboxylate groups of BDC ligand. These free oxygens act as anchoring sites for the metal ions of the metal precursors. Chemical and physical characteristics of the NPs within the framework have been evaluated by the high resolution transmission electron microscopic (HRTEM) images. Excluding Ag(0) and Pd(0) other NPs are present as combinations of their elemental as well as oxide forms (Au/Au 2 O 3 , Cr/Cr 2 O 3 /CrO 2 and Cu/Cu 2 O, Fe/FeO). Synthesized Ag NPs within the framework show remarkable antibacterial efficacy at extremely low concentrations. Ag, Au and Cu/Fe NPs show ferromagnetic properties within the framework at room temperature. This polymer has potential to sequester highly toxic Cr(VI) to non toxic Cr(0), Cr(III) and Cr(IV) species.

Synthesis, characterization, and application of novel Zn(II)-ionic imprinted polymer for preconcentration of Zn(II) ions from aqueous solution

NASA Astrophysics Data System (ADS)

Wirawan, T.; Supriyanto, G.; Soegianto, A.

2018-04-01

Preparation of a new Zn2+ ion-imprinted polymer (Zn-IIP) is presented in this report. The Zn-IIP are prepared by precipitation polymerization using 8-hydroxyquinoline (8HQ) as a ligand, methacrylic acid (MAA) as functional monomer, and ethylene glycol dimethacrylate (EGDMA) as a cross-linker has been prepared. The benzoyl peroxide and ethanol/acetonitrile (2:1) mixture were used as initiator and porogen, respectively. Precipitation polymerization was carried out by heating in a water bath at 60°C for 8 hours. After polymerization, cavities in the polymer particles corresponding to the Zn2+ ions were created by leaching the polymer with 2 mol L-1 HNO3. The polymer was washed with aquabidest and dried in an oven at the temperature of 60°C for 24 hours. The Zn-IIP was characterized by Fourier Transform Infrared Spectrophotometry (FT-IR) and Scanning Electron Microscopy (SEM). The synthesized Zn-IIP was used as a new adsorbent for solid phase extraction (SPE) of Zn(II) prior to Flame Atomic Absorption Spectrometry (FAAS) determination. The experimental parameters for SPE, such as pH of the sample, loading rate, and elution volume, have been optimized. The effect of pH of the sample on the extraction of analyte was studied in batch mode. The effects of loading rate and elution volume on the extraction of analyte were studied in dynamic mode by loading of the sample through IIP-SPE cartridge containing 100 mg of the synthesized Zn-IIP. The imprinted polymer (Zn-IIP) have bands at 3433.06 cm-1 (O-H), 1508.23 cm-1 (C=N aromatics), 1284.5 cm-1 (C-N aromatics), 1056.9 cm-1 (C-O phenol), 1724.24 cm-1 (C=O), and 1639.38 cm-1 (conjugated C=O with C=C). The Scanning Electron Microscopy (SEM) images of IIP and IIP show that the IIP is seen to have more cavities than NIP. The optimum pH for quantitative Zn(II) retention was 5.5, and the elution was completed with 2 mL of 1.0 mol L-1 nitric acid. The optimum loading rate was 0.5 mL min-1. The recovery of Zn(II) from solution

Two new luminescent Zn(II) compounds constructed from guanazole and aromatic polycarboxylate ligands

NASA Astrophysics Data System (ADS)

Zhao, Haixiang; Dong, Yanli; Liu, Haiping

2016-02-01

Two new Zn(II) compounds, namely [(CH3)2NH2]2n[Zn3(bpt)2(datrz)2]n (1) and [(CH3)2NH2)]n[Zn2(bptc)(datrz)]n·n(H2O) (2) (H3bpt = biphenyl-3,4‧,5-tricarboxylic acid, H4bptc = biphenyl-3,3‧,5,5‧-tetracarboxylic acid, Hdatrz = 3,5-diamino-1,2,4-triazole), have been obtained by the self-assemble reactions of Zn(NO3)2, 3,5-diamino-1,2,4-triazole, aromatic polycarboxylate ligands under solvothermal conditions. Single crystal X-ray structural analyses reveal that both compounds display three-dimensional (3D) frameworks. Compound 1 features a trinodal (3, 4, 6)-connected topological network with the point symbol of {4.62}2{4.64.8}{46.64.85}. Compound 2 displays a binodal (4, 6)-connected topological network with the point symbol of {32.62.72}{34.42.64.75}. In addition, the thermal stabilities and luminescent properties of compounds 1 and 2 were also investigated in the solid state at room temperature.

The Cutting Edge of Affinity Electrophoresis Technology

PubMed Central

Kinoshita, Eiji; Kinoshita-Kikuta, Emiko; Koike, Tohru

2015-01-01

Affinity electrophoresis is an important technique that is widely used to separate and analyze biomolecules in the fields of biology and medicine. Both quantitative and qualitative information can be gained through affinity electrophoresis. Affinity electrophoresis can be applied through a variety of strategies, such as mobility shift electrophoresis, charge shift electrophoresis or capillary affinity electrophoresis. These strategies are based on changes in the electrophoretic patterns of biological macromolecules that result from interactions or complex-formation processes that induce changes in the size or total charge of the molecules. Nucleic acid fragments can be characterized through their affinity to other molecules, for example transcriptional factor proteins. Hydrophobic membrane proteins can be identified by means of a shift in the mobility induced by a charged detergent. The various strategies have also been used in the estimation of association/disassociation constants. Some of these strategies have similarities to affinity chromatography, in that they use a probe or ligand immobilized on a supported matrix for electrophoresis. Such methods have recently contributed to profiling of major posttranslational modifications of proteins, such as glycosylation or phosphorylation. Here, we describe advances in analytical techniques involving affinity electrophoresis that have appeared during the last five years. PMID:28248262

Affinity Crystallography: A New Approach to Extracting High-Affinity Enzyme Inhibitors from Natural Extracts.

PubMed

Aguda, Adeleke H; Lavallee, Vincent; Cheng, Ping; Bott, Tina M; Meimetis, Labros G; Law, Simon; Nguyen, Nham T; Williams, David E; Kaleta, Jadwiga; Villanueva, Ivan; Davies, Julian; Andersen, Raymond J; Brayer, Gary D; Brömme, Dieter

2016-08-26

Natural products are an important source of novel drug scaffolds. The highly variable and unpredictable timelines associated with isolating novel compounds and elucidating their structures have led to the demise of exploring natural product extract libraries in drug discovery programs. Here we introduce affinity crystallography as a new methodology that significantly shortens the time of the hit to active structure cycle in bioactive natural product discovery research. This affinity crystallography approach is illustrated by using semipure fractions of an actinomycetes culture extract to isolate and identify a cathepsin K inhibitor and to compare the outcome with the traditional assay-guided purification/structural analysis approach. The traditional approach resulted in the identification of the known inhibitor antipain (1) and its new but lower potency dehydration product 2, while the affinity crystallography approach led to the identification of a new high-affinity inhibitor named lichostatinal (3). The structure and potency of lichostatinal (3) was verified by total synthesis and kinetic characterization. To the best of our knowledge, this is the first example of isolating and characterizing a potent enzyme inhibitor from a partially purified crude natural product extract using a protein crystallographic approach.

Highly selectively monitoring heavy and transition metal ions by a fluorescent sensor based on dipeptide.

PubMed

Neupane, Lok Nath; Thirupathi, Ponnaboina; Jang, Sujung; Jang, Min Jung; Kim, Jung Hwa; Lee, Keun-Hyeung

2011-09-15

Fluorescent sensor (DMH) based on dipeptide was efficiently synthesized in solid phase synthesis. The dipeptide sensor shows sensitive response to Ag(I), Hg(II), and Cu(II) among 14 metal ions in 100% aqueous solution. The fluorescent sensor differentiates three heavy metal ions by response type; turn on response to Ag(I), ratiometric response to Hg(II), and turn off detection of Cu(II). The detection limits of the sensor for Ag(I) and Cu(II) were much lower than the EPA's drinking water maximum contaminant levels (MCL). Specially, DMH penetrated live cells and detected intracellular Ag(+) by turn on response. We described the fluorescent change, binding affinity, detection limit for the metal ions. The study of a heavy metal-responsive sensor based on dipeptide demonstrates its potential utility in the environment field. Copyright © 2011 Elsevier B.V. All rights reserved.

Biophysical and computational comparison on the binding affinity of three important nutrients to β-lactoglobulin: folic acid, ascorbic acid and vitamin K3.

PubMed

Shahraki, Somaye; Heydari, Ali; Saeidifar, Maryam; Gomroki, Masoumeh

2017-11-06

Small globular protein, β-lactoglobulin (βLG), which has significant affinity toward many drugs, is the most abundant whey protein in milk. In this study, the interaction of βLG with three important nutrients, ascorbic acid (ASC), folic acid (FOL), and vitamin K3 (VK3) was investigated by spectroscopic methods (UV-visible and fluorescence) along with molecular docking technique. The results of fluorescence measurements showed that studied nutrients strongly quenched βLG fluorescence in static (FOL and ACS) or static-dynamic combined quenching (VK3) mode. The values of binding constants (K βLG-ASC  ~ 4.34 × 10 4  M -1 , K βLG-FOL ~ 1.67 × 10 4  M -1 and K βLG-VK3 ~ 13.49 × 10 4  M -1 at 310 K) suggested that VK3 and FOL had stronger binding affinity toward βLG than ASC. Thermodynamic analysis indicated that hydrophobic interactions are the major forces in the stability of FOL-βLG complex with enthalpy- and entropy-driving mode while, hydrogen bonds and van der Waals interactions play a major role for βLG-ASC and βLG-VK3 associations. The results of 3D fluorescence FT-IR and UV-Visible measurements indicated that the binding of above nutrients to βLG may induce conformational and micro-environmental changes of protein. Also, there is a reciprocal complement between spectroscopic techniques and molecular docking modeling. The docking results indicate that the ASC, FOL, and VK3 bind to residues located in the subdomain B of βLG. Finally, this report suggests that βLG could be used as an effective carrier of above nutrients in functional foods.

Affinity Spaces and 21st Century Learning

ERIC Educational Resources Information Center

Gee, James Paul

2017-01-01

This article discusses video games as "attractors" to "affinity spaces." It argues that affinity spaces are key sites today where people teach and learn 21st Century skills. While affinity spaces are proliferating on the Internet as interest-and-passion-driven sites devoted to a common set of endeavors, they are not new, just…

Hybrid Integrated Silicon Microfluidic Platform for Fluorescence Based Biodetection.

PubMed

Chandrasekaran, Arvind; Acharya, Ashwin; You, Jian Liang; Soo, Kim Young; Packirisamy, Muthukumaran; Stiharu, Ion; Darveau, André

2007-09-11

The desideratum to develop a fully integrated Lab-on-a-chip device capable ofrapid specimen detection for high throughput in-situ biomedical diagnoses and Point-of-Care testing applications has called for the integration of some of the novel technologiessuch as the microfluidics, microphotonics, immunoproteomics and Micro ElectroMechanical Systems (MEMS). In the present work, a silicon based microfluidic device hasbeen developed for carrying out fluorescence based immunoassay. By hybrid attachment ofthe microfluidic device with a Spectrometer-on-chip, the feasibility of synthesizing anintegrated Lab-on-a-chip type device for fluorescence based biosensing has beendemonstrated. Biodetection using the microfluidic device has been carried out usingantigen sheep IgG and Alexafluor-647 tagged antibody particles and the experimentalresults prove that silicon is a compatible material for the present application given thevarious advantages it offers such as cost-effectiveness, ease of bulk microfabrication,superior surface affinity to biomolecules, ease of disposability of the device etc., and is thussuitable for fabricating Lab-on-a-chip type devices.

On the structure of self-affine convex bodies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Voynov, A S

2013-08-31

We study the structure of convex bodies in R{sup d} that can be represented as a union of their affine images with no common interior points. Such bodies are called self-affine. Vallet's conjecture on the structure of self-affine bodies was proved for d = 2 by Richter in 2011. In the present paper we disprove the conjecture for all d≥3 and derive a detailed description of self-affine bodies in R{sup 3}. Also we consider the relation between properties of self-affine bodies and functional equations with a contraction of an argument. Bibliography: 10 titles.

Tryptophan W207 in transducin T alpha is the fluorescence sensor of the G protein activation switch and is involved in the effector binding.

PubMed Central

Faurobert, E; Otto-Bruc, A; Chardin, P; Chabre, M

1993-01-01

We have produced a recombinant transducin alpha subunit (rT alpha) in sf9 cells, using a baculovirus system. Deletion of the myristoylation site near the N-terminal increased the solubility and allowed the purification of rT alpha. When reconstituted with excess T beta gamma on retinal membrane, rT alpha displayed functional characteristics of wild-type T alpha vis à vis its coupled receptor, rhodopsin and its effector, cGMP phosphodiesterase (PDE). We further mutated a tryptophan, W207, which is conserved in all G proteins and is suspected to elicit the fluorescence change correlated to their activation upon GDP/GTP exchange or aluminofluoride (AlFx) binding. [W207F]T alpha mutant displayed high affinity receptor binding and underwent a conformational switch upon receptor-catalysed GTP gamma S binding or upon AlFx binding, but this did not elicit any fluorescence change. Thus W207 is the only fluorescence sensor of the switch. Upon the switch the mutant remained unable to activate the PDE. To characterize better its effector-activating interaction we measured the affinity of [W207F]T alpha GDP-AlFx for PDE gamma, the effector subunit that binds most tightly to T alpha. [W207F]T alpha still bound in an activation-dependent way to PDE gamma, but with a 100-fold lower affinity than rT alpha. This suggests that W207 contributes to the G protein effector binding. Images PMID:8223434

Improved Tumor Targeting and Longer Retention Time of NIR Fluorescent Probes Using Bioorthogonal Chemistry.

PubMed

Zhang, Xianghan; Wang, Bo; Zhao, Na; Tian, Zuhong; Dai, Yunpeng; Nie, Yongzhan; Tian, Jie; Wang, Zhongliang; Chen, Xiaoyuan

2017-01-01

The traditional labeling method for targeted NIR fluorescence probes requires directly covalent-bonded conjugation of targeting domains and fluorophores in vitro . Although this strategy works well, it is not sufficient for detecting or treating cancers in vivo , due to steric hindrance effects that relatively large fluorophore molecules exert on the configurations and physiological functions of specific targeting domains. The copper-free, "click-chemistry"-assisted assembly of small molecules in living systems may enhance tumor accumulation of fluorescence probes by improving the binding affinities of the targeting factors. Here, we employed a vascular homing peptide, GEBP11, as a targeting factor for gastric tumors, and we demonstrate its effectiveness for in vivo imaging via click-chemistry-mediated conjugation with fluorescence molecules in tumor xenograft mouse models. This strategy showed higher binding affinities than those of the traditional conjugation method, and our results showed that the tumor accumulation of click-chemistry-mediated probes are 11-fold higher than that of directly labeled probes. The tracking life was prolonged by 12-fold, and uptake of the probes into the kidney was reduced by 6.5-fold. For lesion tumors of different sizes, click-chemistry-mediated probes can achieve sufficient signal-to-background ratios (3.5-5) for in vivo detection, and with diagnostic sensitivity approximately 3.5 times that of traditional labeling probes. The click-chemistry-assisted detection strategy utilizes the advantages of "small molecule" probes while not perturbing their physiological functions; this enables tumor detection with high sensitivity and specific selectivity.

A nitrogen-dependent switch in the high affinity ammonium transport in Medicago truncatula.

PubMed

Straub, Daniel; Ludewig, Uwe; Neuhäuser, Benjamin

2014-11-01

Ammonium transporters (AMTs) are crucial for the high affinity primary uptake and translocation of ammonium in plants. In the model legume Medicago truncatula, the genomic set of AMT-type ammonium transporters comprises eight members. Only four genes were abundantly expressed in young seedlings, both in roots and shoots. While the expression of all AMTs in the shoot was not affected by the nitrogen availability, the dominating MtAMT1;1 gene was repressed by nitrogen in roots, despite that cellular nitrogen concentrations were far above deficiency levels. A contrasting de-repression by nitrogen was observed for MtAMT1;4 and MtAMT2;1, which were both expressed at intermediate level. Weak expression was found for MtAMT1;2 and MtAMT2;3, while the other AMTs were not detected in young seedlings. When expressed from their endogenous promoters, translational fusion proteins of MtAMT1;1 and MtAMT2;1 with green fluorescent protein were co-localized in the plasma membrane of rhizodermal cells, but also detected in cortical root layers. Both transporter proteins similarly functionally complemented a yeast strain that is deficient in high affinity ammonium transport, both at acidic and neutral pH. The uptake into yeast mediated by these transporters saturated with Km AMT1;1 = 89 µM and Km AMT2;1 = 123 µM, respectively. When expressed in oocytes, MtAMT1;1 mediated much larger (15)N-ammonium uptake than MtAMT2;1, but NH4 (+) currents were only recorded for MtAMT1;1. These currents saturated with a voltage-dependent Km = 90 µM at -80 mV. The cellular localization and regulation of the AMTs suggests that MtAMT1;1 encodes the major high affinity ammonium transporter gene in low nitrogen grown young M. truncatula roots and despite the similar localization and substrate affinity, MtAMT2;1 appears functionally distinct and more important at higher nitrogen supply.

Pattern-based detection of anion pollutants in water with DNA polyfluorophores† †Electronic supplementary information (ESI) available: Additional figures and tables and experimental details. See DOI: 10.1039/c4sc03992k Click here for additional data file.

PubMed Central

Kwon, Hyukin; Jiang, Wei

2015-01-01

Many existing irrigation, industrial and chemical storage sites are currently introducing hazardous anions into groundwater, making the monitoring of such sites a high priority. Detecting and quantifying anions in water samples typically requires complex instrumentation, adding cost and delaying analysis. Here we address these challenges by development of an optical molecular method to detect and discriminate a broad range of anionic contaminants with DNA-based fluorescent sensors. A library of 1296 tetrameric-length oligodeoxyfluorosides (ODFs) composed of metal ligand and fluorescence modulating monomers was constructed with a DNA synthesizer on PEG-polystyrene microbeads. These oligomers on beads were incubated with YIII or ZnII ions to provide affinity and responsiveness to anions. Seventeen anions were screened with the library under an epifluorescence microscope, ultimately yielding eight chemosensors that could discriminate 250 μM solutions of all 17 anions in buffered water using their patterns of response. This sensor set was able to identify two unknown anion samples from ten closely-responding anions and could also function quantitatively, determining unknown concentrations of anions such as cyanide (as low as 1 mM) and selenate (as low as 50 μM). Further studies with calibration curves established detection limits of selected anions including thiocyanate (detection limit ∼300 μM) and arsenate (∼800 μM). The results demonstrate DNA-like fluorescent chemosensors as versatile tools for optically analyzing environmentally hazardous anions in aqueous environments. PMID:26146537

Kojic acid biosynthesis in Aspergillus oryzae is regulated by a Zn(II)(2)Cys(6) transcriptional activator and induced by kojic acid at the transcriptional level.

PubMed

Marui, Junichiro; Yamane, Noriko; Ohashi-Kunihiro, Sumiko; Ando, Tomohiro; Terabayashi, Yasunobu; Sano, Motoaki; Ohashi, Shinichi; Ohshima, Eiji; Tachibana, Kuniharu; Higa, Yoshitaka; Nishimura, Marie; Koike, Hideaki; Machida, Masayuki

2011-07-01

A gene encoding the Zn(II)(2)Cys(6) transcriptional factor is clustered with two genes involved in biosynthesis of a secondary metabolite, kojic acid (KA), in Aspergillus oryzae. We determined that the gene was essential for KA production and the transcriptional activation of KA biosynthetic genes, which were triggered by the addition of KA. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Classical affine W-algebras associated to Lie superalgebras

NASA Astrophysics Data System (ADS)

Suh, Uhi Rinn

2016-02-01

In this paper, we prove classical affine W-algebras associated to Lie superalgebras (W-superalgebras), which can be constructed in two different ways: via affine classical Hamiltonian reductions and via taking quasi-classical limits of quantum affine W-superalgebras. Also, we show that a classical finite W-superalgebra can be obtained by a Zhu algebra of a classical affine W-superalgebra. Using the definition by Hamiltonian reductions, we find free generators of a classical W-superalgebra associated to a minimal nilpotent. Moreover, we compute generators of the classical W-algebra associated to spo(2|3) and its principal nilpotent. In the last part of this paper, we introduce a generalization of classical affine W-superalgebras called classical affine fractional W-superalgebras. We show these have Poisson vertex algebra structures and find generators of a fractional W-superalgebra associated to a minimal nilpotent.

Application of fluorescently labeled tracer technique for detection of natural active macromolecules in Chinese medicine.

PubMed

Zeng, Qiao-Hui; Zhang, Xue-Wu; Xu, Kai-Peng; Jiang, Jian-Guo

2014-02-01

Active substances in traditional Chinese Medicine (TCM) contain not only a variety of small molecules, but also many other macromolecules (TCMMs), such as proteins, peptides and polysaccharides. Active TCMM can achieve good therapeutic effects by regulating the body's overall function with lower side effects. This review summarized the literatures published in recent years on the application of fluorescently labeled tracer technique for detection of natural active macromolecules in TCM. Classified by fluorescent markers, applications of fluorescein, rhodamine, and quantum dots (QDs) in TCMM active tracer are reviewed, and the methods and principles of TCMM fluorescent marker are illustrated. Studies on active TCMMs and their action mechanism are quite difficult due to a multitarget, multicomponent, and multipath system of TCM. However, the development of fluorescently labeled active tracer technique (FLATT) provides this research with new tools. Traditional fluorescent markers have many deficiencies, such as easily quenched, short luminous cycle, and intrinsic toxicity. Relatively, FLATT has many obvious advantages, and its application in TCMM is still at the early stage. In order to improve the overall level of fluorescence labeling in TCMM active tracer, the improvement on FLATT's detection sensitivity and biological affinity is urgent and critical to allow study of these interesting molecules.

A small-molecule-linked DNA-graphene oxide-based fluorescence-sensing system for detection of biotin.

PubMed

Zhang, Hao; Li, Yan; Su, Xingguang

2013-11-15

In this paper, we establish a novel fluorescence-sensing system for the detection of biotin based on the interaction between DNA and graphene oxide and on protection of the terminal of the biotinylated single-stranded DNA fluorescent probe by streptavidin. In this system, streptavidin binds to the biotinylated DNA, which protects the DNA from hydrolysis by exonuclease I. The streptavidin-DNA conjugate is then adsorbed to the graphene oxide resulting in the fluorescence being quenched. Upon the addition of free biotin, it competes with the labeled biotin for the binding sites of streptavidin and then the exonuclease I digests the unbound DNA probe from the 3' to the 5' terminal, releasing the fluorophore from the DNA. Because of the weak affinity between the fluorophore and graphene oxide, the fluorescence is recovered. Under optimal conditions, the fluorescence intensity is proportional to the concentration of biotin in the concentration range of 0.5-20nmol/L. The detection limit for biotin is 0.44nmol/L. The proposed fluorescence-sensing system was applied to the determination of biotin in some real samples with satisfactory reproducibility and accuracy. This work could provide a common platform for detecting small biomolecules based on protein-small molecule ligand binding. Copyright © 2013 Elsevier Inc. All rights reserved.

Semiconductor Nanomaterials-Based Fluorescence Spectroscopic and Matrix-Assisted Laser Desorption/Ionization (MALDI) Mass Spectrometric Approaches to Proteome Analysis

PubMed Central

Kailasa, Suresh Kumar; Cheng, Kuang-Hung; Wu, Hui-Fen

2013-01-01

Semiconductor quantum dots (QDs) or nanoparticles (NPs) exhibit very unusual physico-chemcial and optical properties. This review article introduces the applications of semiconductor nanomaterials (NMs) in fluorescence spectroscopy and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for biomolecule analysis. Due to their unique physico-chemical and optical properties, semiconductors NMs have created many new platforms for investigating biomolecular structures and information in modern biology. These semiconductor NMs served as effective fluorescent probes for sensing proteins and cells and acted as affinity or concentrating probes for enriching peptides, proteins and bacteria proteins prior to MALDI-MS analysis. PMID:28788422

Peptide aptamer-assisted immobilization of green fluorescent protein for creating biomolecule-complexed carbon nanotube device

NASA Astrophysics Data System (ADS)

Nii, Daisuke; Nozawa, Yosuke; Miyachi, Mariko; Yamanoi, Yoshinori; Nishihara, Hiroshi; Tomo, Tatsuya; Shimada, Yuichiro

2017-10-01

Carbon nanotubes are a novel material for next-generation applications. In this study, we generated carbon nanotube and green fluorescent protein (GFP) conjugates using affinity binding peptides. The carbon nanotube-binding motif was introduced into the N-terminus of the GFP through molecular biology methods. Multiple GFPs were successfully aligned on a single-walled carbon nanotube via the molecular recognition function of the peptide aptamer, which was confirmed through transmission electron microscopy and optical analysis. Fluorescence spectral analysis results also suggested that the carbon nanotube-GFP complex was autonomously formed with orientation and without causing protein denaturation during immobilization. This simple process has a widespread potential for fabricating carbon nanotube-biomolecule hybrid devices.

Amyloid-β Deposits Target Efficient Near-Infrared Fluorescent Probes: Synthesis, in Vitro Evaluation, and in Vivo Imaging.

PubMed

Fu, Hualong; Tu, Peiyu; Zhao, Liu; Dai, Jiapei; Liu, Boli; Cui, Mengchao

2016-02-02

The formation of extracellular amyloid-β (Aβ) plaques is a common molecular change that underlies several debilitating human conditions, including Alzheimer's disease (AD); however, the existing near-infrared (NIR) fluorescent probes for the in vivo detection of Aβ plaques are limited by undesirable fluorescent properties and poor brain kinetics. In this work, we designed, synthesized, and evaluated a new family of efficient NIR probes that target Aβ plaques by incorporating hydroxyethyl groups into the ligand structure. Among these probes, DANIR 8c showed excellent fluorescent properties with an emission maximum above 670 nm upon binding to Aβ aggregates and also displayed a high sensitivity (a 629-fold increase in fluorescence intensity) and affinity (Kd = 14.5 nM). Because of the improved hydrophilicity that was induced by hydroxyls, 8c displayed increased initial brain uptake and a fast washout from the brain, as well as an acceptable biostability in the brain. In vivo NIR fluorescent imaging revealed that 8c could efficiently distinguish between AD transgenic model mice and normal controls. Overall, 8c is an efficient and veritable NIR fluorescent probe for the in vivo detection of Aβ plaques in the brain.

Differential structural properties of GLP-1 and exendin-4 determine their relative affinity for the GLP-1 receptor N-terminal extracellular domain.

PubMed

Runge, Steffen; Schimmer, Susann; Oschmann, Jan; Schiødt, Christine Bruun; Knudsen, Sanne Möller; Jeppesen, Claus Bekker; Madsen, Kjeld; Lau, Jesper; Thøgersen, Henning; Rudolph, Rainer

2007-05-15

Glucagon-like peptide-1 (GLP-1) and exendin-4 (Ex4) are homologous peptides with established potential for treatment of type 2 diabetes. They bind and activate the pancreatic GLP-1 receptor (GLP-1R) with similar affinity and potency and thereby promote insulin secretion in a glucose-dependent manner. GLP-1R belongs to family B of the seven transmembrane G-protein coupled receptors. The N-terminal extracellular domain (nGLP-1R) is a ligand binding domain with differential affinity for Ex4 and GLP-1: low affinity for GLP-1 and high affinity for exendin-4. The superior affinity of nGLP-1R for Ex4 was previously explained by an additional interaction between nGLP-1R and the C-terminal Trp-cage of Ex4. In this study we have combined biophysical and pharmacological approaches thus relating structural properties of the ligands in solution to their relative binding affinity for nGLP-1R. We used both a tracer competition assay and ligand-induced thermal stabilization of nGLP-1R to measure the relative affinity of full length, truncated, and chimeric ligands for soluble refolded nGLP-1R. The ligands in solution and the conformational consequences of ligand binding to nGLP-1R were characterized by circular dichroism and fluorescence spectroscopy. We found a correlation between the helical content of the free ligands and their relative binding affinity for nGLP-1R, supporting the hypothesis that the ligands are helical at least in the segment that binds to nGLP-1R. The Trp-cage of Ex4 was not necessary to maintain a superior helicity of Ex4 compared to GLP-1. The results suggest that the differential affinity of nGLP-1R is explained almost entirely by divergent residues in the central part of the ligands: Leu10-Gly30 of Ex4 and Val16-Arg36 of GLP-1. In view of our results it appears that the Trp-cage plays only a minor role for the interaction between Ex4 and nGLP-1R and for the differential affinity of nGLP-1R for GLP-1 and Ex4.

Classical affine W-algebras associated to Lie superalgebras

DOE Office of Scientific and Technical Information (OSTI.GOV)

Suh, Uhi Rinn, E-mail: uhrisu1@math.snu.ac.kr

2016-02-15

In this paper, we prove classical affine W-algebras associated to Lie superalgebras (W-superalgebras), which can be constructed in two different ways: via affine classical Hamiltonian reductions and via taking quasi-classical limits of quantum affine W-superalgebras. Also, we show that a classical finite W-superalgebra can be obtained by a Zhu algebra of a classical affine W-superalgebra. Using the definition by Hamiltonian reductions, we find free generators of a classical W-superalgebra associated to a minimal nilpotent. Moreover, we compute generators of the classical W-algebra associated to spo(2|3) and its principal nilpotent. In the last part of this paper, we introduce a generalizationmore » of classical affine W-superalgebras called classical affine fractional W-superalgebras. We show these have Poisson vertex algebra structures and find generators of a fractional W-superalgebra associated to a minimal nilpotent.« less

Tryptophan tags and de novo designed complementary affinity ligands for the expression and purification of recombinant proteins.

PubMed

Pina, Ana Sofia; Carvalho, Sara; Dias, Ana Margarida G C; Guilherme, Márcia; Pereira, Alice S; Caraça, Luciana T; Coroadinha, Ana Sofia; Lowe, Christopher R; Roque, A Cecília A

2016-11-11

A common strategy for the production and purification of recombinant proteins is to fuse a tag to the protein terminal residues and employ a "tag-specific" ligand for fusion protein capture and purification. In this work, we explored the effect of two tryptophan-based tags, NWNWNW and WFWFWF, on the expression and purification of Green Fluorescence Protein (GFP) used as a model fusion protein. The titers obtained with the expression of these fusion proteins in soluble form were 0.11mgml -1 and 0.48mgml -1 for WFWFWF and NWNWNW, respectively. A combinatorial library comprising 64 ligands based on the Ugi reaction was prepared and screened for binding GFP-tagged and non-tagged proteins. Complementary ligands A2C2 and A3C1 were selected for the effective capture of NWNWNW and WFWFWF tagged proteins, respectively, in soluble forms. These affinity pairs displayed 10 6 M -1 affinity constants and Qmax values of 19.11±2.60ugg -1 and 79.39ugg -1 for the systems WFWFWF AND NWNWNW, respectively. GFP fused to the WFWFWF affinity tag was also produced as inclusion bodies, and a refolding-on column strategy was explored using the ligand A4C8, selected from the combinatorial library of ligands but in presence of denaturant agents. Copyright © 2016 Elsevier B.V. All rights reserved.

Alternative Affinity Ligands for Immunoglobulins.

PubMed

Kruljec, Nika; Bratkovič, Tomaž

2017-08-16

The demand for recombinant therapeutic antibodies and Fc-fusion proteins is expected to increase in the years to come. Hence, extensive efforts are concentrated on improving the downstream processing. In particular, the development of better-affinity chromatography matrices, supporting robust time- and cost-effective antibody purification, is warranted. With the advances in molecular design and high-throughput screening approaches from chemical and biological combinatorial libraries, novel affinity ligands representing alternatives to bacterial immunoglobulin (Ig)-binding proteins have entered the scene. Here, we review the design, development, and properties of diverse classes of alternative antibody-binding ligands, ranging from engineered versions of Ig-binding proteins, to artificial binding proteins, peptides, aptamers, and synthetic small-molecular-weight compounds. We also provide examples of applications for the novel affinity matrices in chromatography and beyond.

Characterization and validation of fluorescent receptor ligands: a case study of the ionotropic serotonin receptor.

PubMed

Hovius, Ruud

2013-01-01

The application of fluorescent receptor ligands has become widespread, incited by two important reasons. "Seeing is believing"-it is possible to visualize in real time in live cells ligand-receptor interactions, and to locate the receptors with subcellular precision allowing one to follow, e.g., internalization of the ligand-receptor complex. The high sensitivity of photon detection permits observation of on the one hand receptor-ligand interactions on cells with low, native receptor abundance, and on the other of individual fluorophores unveiling the stochastic properties of single ligand-receptor complexes.The major bottlenecks that impede extensive use of fluorescent ligands are due to possible dramatic changes of the pharmacological properties of a ligand upon chemical modification and fluorophore conjugation, aggravated by the observation that different fluorophores can provoke very dissimilar effects. This makes it virtually impossible to predict beforehand which labelling strategy to use to produce a fluorescent ligand with the desired qualities.Here, we focus on the design, synthesis, and evaluation of a high-affinity fluorescent antagonist for the ionotropic serotonin type-3 receptor.

A fluorescent aptasensor based on a DNA pyramid nanostructure for ultrasensitive detection of ochratoxin A.

PubMed

Nameghi, Morteza Alinezhad; Danesh, Noor Mohammad; Ramezani, Mohammad; Hassani, Faezeh Vahdati; Abnous, Khalil; Taghdisi, Seyed Mohammad

2016-08-01

Analytical techniques for detection of ochratoxin A (OTA) in food products and blood serum are of great significance. In this study, a fluorescent aptasensor was developed for sensitive and specific detection of OTA, based on a DNA pyramid nanostructure (DPN) and PicoGreen (PG) dye. The designed aptasensor inherits characteristics of DPN, such as high stability and capacity for PG loading. PG, as a fluorescent dye, could bind to double-stranded DNA (dsDNA). In the absence of OTA, the pyramid structure of DPN remains intact, leading to a very strong fluorescence emission. Because of higher affinity of aptamer for its target relative to its complementary strand, upon addition of target, the pyramid structure of DPN is disassembled, leading to a weak fluorescence emission. The presented aptasensor showed high specificity toward OTA with a limit of detection (LOD) as low as 0.135 nM. Besides, the designed sensing strategy was successfully utilized to recognize OTA in serum and grape juice with LODs of 0.184 and 0.149 nM, respectively.

Selection of imprinted nanoparticles by affinity chromatography.

PubMed

Guerreiro, António R; Chianella, Iva; Piletska, Elena; Whitcombe, Michael J; Piletsky, Sergey A

2009-04-15

Soluble molecularly imprinted nanoparticles were synthesised via iniferter initiated polymerisation and separated by size via gel permeation chromatography. Subsequent fractionation of these particles by affinity chromatography allowed the separation of high affinity fractions from the mixture of nanoparticles. Fractions selected this way possess affinity similar to that of natural antibodies (K(d) 6.6x10(-8)) M and were also able to discriminate between related functional analogues of the template.

Specific capture and detection of Staphylococcus aureus with high-affinity modified aptamers to cell surface components.

PubMed

Baumstummler, A; Lehmann, D; Janjic, N; Ochsner, U A

2014-10-01

Slow off-rate modified aptamer (SOMAmer) reagents were generated to several Staphylococcus aureus cell surface-associated proteins via SELEX with multiple modified DNA libraries using purified recombinant or native proteins. High-affinity binding agents with sub-nanomolar Kd 's were obtained for staphylococcal protein A (SpA), clumping factors (ClfA, ClfB), fibronectin-binding proteins (FnbA, FnbB) and iron-regulated surface determinants (Isd). Further screening revealed several SOMAmers that specifically bound to Staph. aureus cells from all strains that were tested, but not to other staphylococci or other bacteria. SpA and ClfA SOMAmers proved useful for the selective capture and enrichment of Staph. aureus cells, as shown by culture and PCR, leading to improved limits of detection and efficient removal of PCR inhibitors. Detection of Staph. aureus cells was enhanced by several orders of magnitude when the bacterial cell surface was coated with SOMAmers followed by qPCR of the SOMAmers. Furthermore, fluorescence-labelled SpA SOMAmers demonstrated their utility as direct detection agents in flow cytometry. Significance and impact of the study: Monitoring for microbial contamination of food, water, nonsterile products or the environment is typically based on culture, PCR or antibodies. Aptamers that bind with high specificity and affinity to well-conserved cell surface epitopes represent a promising novel type of reagents to detect bacterial cells without the need for culture or cell lysis, including for the capture and enrichment of bacteria present at low cell densities and for the direct detection via qPCR or fluorescent staining. © 2014 Soma Logic, Inc. published by John Wiley & Sons Ltd On behalf of the society for Applied Microbiology.

Vaccine Efficacy and Affinity Maturation

NASA Astrophysics Data System (ADS)

Lee, Hayoun; Deem, Michael W.

2002-03-01

We propose macroscopic equations to describe variable vaccine efficacy between repeated vaccinee and first time vaccinee. The main ingredients are antigenic distance between epidemic strain and vaccne strain, and affinity maturation dynamics which differs in primary and second response. Increase of affinity by repeated vaccine leads to localization in immune space. This localization decreases the ability of the immune system to response to distant, but related epidemic strains.

DNA-hosted copper nanoclusters/graphene oxide based fluorescent biosensor for protein kinase activity detection.

PubMed

Wang, Mengke; Lin, Zihan; Liu, Qing; Jiang, Shan; Liu, Hua; Su, Xingguang

2018-07-05

A novel fluorescent biosensor for protein kinase activity (PKA) detection was designed by applying double-strands DNA-hosted copper nanoclusters (dsDNA-CuNCs) and graphene oxide (GO). One DNA strand of the dsDNA consisted of two domains, one domain can hybridize with another complementary DNA strand to stabilize the fluorescent CuNCs and another domain was adenosine 5'-triphosphate (ATP) aptamer. ATP aptamer of the dsDNA-CuNCs would be spontaneously absorbed onto the GO surface through π-π stacking interactions. Thus GO can efficiently quench the fluorescence (FL) of dsDNA-CuNCs through fluorescence resonance energy transfer (FRET). In the present of ATP, ATP specifically combined with ATP aptamer to form ATP-ATP aptamer binding complexes, which had much less affinity to GO, resulting in the fluorescence recovery of the system. Nevertheless, in the presence of PKA, ATP could be translated into ADP and ADP could not combine with ATP aptamer resulting in the fluorescence quenching of dsDNA-CuNCs again. According to the change of the fluorescence signal, PKA activity could be successfully monitored in the range of 0.1-5.0 U mL -1 with a detection limit (LOD) of 0.039 U mL -1 . Besides, the inhibitory effect of H-89 on PKA activity was studied. The sensor was performed for PKA activity detection in cell lysates with satisfactory results. Copyright © 2018 Elsevier B.V. All rights reserved.

β-Lactam Antibiotics with a High Affinity for PBP2 Act Synergistically with the FtsZ-Targeting Agent TXA707 against Methicillin-Resistant Staphylococcus aureus

PubMed Central

Ferrer-González, Edgar; Kaul, Malvika; Parhi, Ajit K.; LaVoie, Edmond J.

2017-01-01

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant pathogen that poses a significant risk to global health today. We have developed a promising new FtsZ-targeting agent (TXA707) with potent activity against MRSA isolates resistant to current standard-of-care antibiotics. We present here results that demonstrate differing extents of synergy between TXA707 and a broad range of β-lactam antibiotics (including six cephalosporins, two penicillins, and two carbapenems) against MRSA. To explore whether there is a correlation between the extent of synergy and the preferential antibacterial target of each β-lactam, we determined the binding affinities of the β-lactam antibiotics for each of the four native penicillin-binding proteins (PBPs) of S. aureus using a fluorescence anisotropy competition assay. A comparison of the resulting PBP binding affinities with our corresponding synergy results reveals that β-lactams with a high affinity for PBP2 afford the greatest degree of synergy with TXA707 against MRSA. In addition, we present fluorescence and electron microscopy studies that suggest a potential mechanism underlying the synergy between TXA707 and the β-lactam antibiotics. In this connection, our microscopy results show a disruption of septum formation in TXA707-treated MRSA cells, with a concomitant mislocalization of the PBPs from midcell to nonproductive peripheral sites. Viewed as a whole, our results indicate that PBP2-targeting β-lactam antibiotics are optimal synergistic partners with FtsZ-targeting agents for use in combination therapy of MRSA infections. PMID:28630190

Controlled intracellular generation of reactive oxygen species in human mesenchymal stem cells using porphyrin conjugated nanoparticles

NASA Astrophysics Data System (ADS)

Lavado, Andrea S.; Chauhan, Veeren M.; Alhaj Zen, Amer; Giuntini, Francesca; Jones, D. Rhodri E.; Boyle, Ross W.; Beeby, Andrew; Chan, Weng C.; Aylott, Jonathan W.

2015-08-01

Nanoparticles capable of generating controlled amounts of intracellular reactive oxygen species (ROS), that advance the study of oxidative stress and cellular communication, were synthesized by functionalizing polyacrylamide nanoparticles with zinc(ii) porphyrin photosensitisers. Controlled ROS production was demonstrated in human mesenchymal stem cells (hMSCs) through (1) production of nanoparticles functionalized with varying percentages of Zn(ii) porphyrin and (2) modulating the number of doses of excitation light to internalized nanoparticles. hMSCs challenged with nanoparticles functionalized with increasing percentages of Zn(ii) porphyrin and high numbers of irradiations of excitation light were found to generate greater amounts of ROS. A novel dye, which is transformed into fluorescent 7-hydroxy-4-trifluoromethyl-coumarin in the presence of hydrogen peroxide, provided an indirect indicator for cumulative ROS production. The mitochondrial membrane potential was monitored to investigate the destructive effect of increased intracellular ROS production. Flow cytometric analysis of nanoparticle treated hMSCs suggested irradiation with excitation light signalled controlled apoptotic cell death, rather than uncontrolled necrotic cell death. Increased intracellular ROS production did not induce phenotypic changes in hMSC subcultures.Nanoparticles capable of generating controlled amounts of intracellular reactive oxygen species (ROS), that advance the study of oxidative stress and cellular communication, were synthesized by functionalizing polyacrylamide nanoparticles with zinc(ii) porphyrin photosensitisers. Controlled ROS production was demonstrated in human mesenchymal stem cells (hMSCs) through (1) production of nanoparticles functionalized with varying percentages of Zn(ii) porphyrin and (2) modulating the number of doses of excitation light to internalized nanoparticles. hMSCs challenged with nanoparticles functionalized with increasing percentages of Zn(ii

Metal Ion Dependence of the Matrix Metalloproteinase-1 Mechanism.

PubMed

Yang, Hao; Makaroff, Katherine; Paz, Nicholas; Aitha, Mahesh; Crowder, Michael W; Tierney, David L

2015-06-16

Matrix metalloproteinase-1 (MMP-1) plays crucial roles in disease-related physiologies and pathological processes in the human body. We report here solution studies of MMP-1, including characterization of a series of mutants designed to bind metal in either the catalytic site or the structural site (but not both). Circular dichroism and fluorescence spectroscopy of the mutants demonstrate the importance of the structural Zn(II) in maintaining both secondary and tertiary structure, while UV-visible, nuclear magnetic resonance, electron paramagnetic resonance, and extended X-ray absorption fine structure show its presence influences the catalytic metal ion's coordination number. The mutants allow us to demonstrate convincingly the preparation of a mixed-metal analogue, Co(C)Zn(S)-MMP-1, with Zn(II) in the structural site and Co(II) in the catalytic site. Stopped-flow fluorescence of the native form, Zn(C)Zn(S)-MMP-1, and the mixed-metal Co(C)Zn(S)-MMP-1 analogue shows that the internal fluorescence of a nearby Trp residue is modulated with catalysis and can be used to monitor reactivity under a number of conditions, opening the door to substrate profiling.

A twice-as-smart synthetic G-quartet: PyroTASQ is both a smart quadruplex ligand and a smart fluorescent probe.

PubMed

Laguerre, Aurélien; Stefan, Loic; Larrouy, Manuel; Genest, David; Novotna, Jana; Pirrotta, Marc; Monchaud, David

2014-09-03

Recent and unambiguous evidences of the formation of DNA and RNA G-quadruplexes in cells has provided solid support for these structures to be considered as valuable targets in oncology. Beyond this, they have lent further credence to the anticancer strategies relying on small molecules that selectively target these higher-order DNA/RNA architectures, referred to as G-quadruplex ligands. They have also shed bright light on the necessity of designing multitasking ligands, displaying not only enticing quadruplex interacting properties (affinity, structural selectivity) but also additional features that make them usable for detecting quadruplexes in living cells, notably for determining whether, when, and where these structures fold and unfold during the cell cycle and also for better assessing the consequences of their stabilization by external agents. Herein, we report a brand new design of such multitasking ligands, whose structure experiences a quadruplex-promoted conformational switch that triggers not only its quadruplex affinity (i.e., smart ligands, which display high affinity and selectivity for DNA/RNA quadruplexes) but also its fluorescence (i.e., smart probes, which behave as selective light-up fluorescent reporters on the basis of a fluorogenic electron redistribution). The first prototype of such multifunctional ligands, termed PyroTASQ, represents a brand new generation of quadruplex ligands that can be referred to as "twice-as-smart" quadruplex ligands.

Synthesis and spectral characterization of Zn(II) microsphere series for antimicrobial application

NASA Astrophysics Data System (ADS)

Singh, Ajay K.; Pandey, Sarvesh K.; Pandey, O. P.; Sengupta, S. K.

2014-09-01

Microsphere series have been synthesized by reacting zinc(II) acetate dihydrate with Schiff bases derived from 2-hydrazino-5-[substituted phenyl]-1,3,4-thiadiazole/oxadiazole/triazole with salicylaldehyde. Elemental analysis suggests that the complexes have 1:2 and 1:1 stoichiometry of the type [Zn(L)2(H2O)2] and [Zn(L‧)(H2O)2]; LH = Schiff bases derived from 2-hydrazino-5-[substituted phenyl]-1,3,4-thia/oxadiazole with salicylaldehyde; L‧H2 = Schiff bases derived from 3-(substituted phenyl)-4-amino-5-hydrazino-1,2,4-triazole and salicylaldehyde and were characterized by elemental analyses, IR, 1H NMR and 13C NMR spectral data. Scanning electron microscopy (SEM) showed that synthesized materials have microsphere like structure and there EDX analysis comparably matches with elemental analysis. For the antimicrobial application Schiff bases and their zinc(II) complexes were screened for four bacteria e.g. Bacillus subtilis, Pseudomonas aeruginosa, Salmonella typhi, Streptococcus pyogenes and four fungi e.g. Cyrtomium falcatum, Aspergillus niger, Fusarium oxysporium and Curvularia pallescence by the reported method. Schiff base and Zn(II) compounds showed significant antimicrobial activities. However, activities increase upon chelation. Thermal analysis (TGA) data of compound (10) showed its stability up to 300 °C.

Sulfated Metabolites of Polychlorinated Biphenyls Are High-Affinity Ligands for the Thyroid Hormone Transport Protein Transthyretin

PubMed Central

Grimm, Fabian A.; Lehmler, Hans-Joachim; He, Xianran; Robertson, Larry W.

2013-01-01

Background: The displacement of l-thyroxine (T4) from binding sites on transthyretin (TTR) is considered a significant contributing mechanism in polychlorinated biphenyl (PCB)-induced thyroid disruption. Previous research has discovered hydroxylated PCB metabolites (OH-PCBs) as high-affinity ligands for TTR, but the binding potential of conjugated PCB metabolites such as PCB sulfates has not been explored. Objectives: We evaluated the binding of five lower-chlorinated PCB sulfates to human TTR and compared their binding characteristics to those determined for their OH-PCB precursors and for T4. Methods: We used fluorescence probe displacement studies and molecular docking simulations to characterize the binding of PCB sulfates to TTR. The stability of PCB sulfates and the reversibility of these interactions were characterized by HPLC analysis of PCB sulfates after their binding to TTR. The ability of OH-PCBs to serve as substrates for human cytosolic sulfotransferase 1A1 (hSULT1A1) was assessed by OH-PCB–dependent formation of adenosine-3´,5´-diphosphate, an end product of the sulfation reaction. Results: All five PCB sulfates were able to bind to the high-affinity binding site of TTR with equilibrium dissociation constants (Kd values) in the low nanomolar range (4.8–16.8 nM), similar to that observed for T4 (4.7 nM). Docking simulations provided corroborating evidence for these binding interactions and indicated multiple high-affinity modes of binding. All OH-PCB precursors for these sulfates were found to be substrates for hSULT1A1. Conclusions: Our findings show that PCB sulfates are high-affinity ligands for human TTR and therefore indicate, for the first time, a potential relevance for these metabolites in PCB-induced thyroid disruption. PMID:23584369

Determining the binding affinities of phenolic compounds to proteins by quenching of the intrinsic tryptophan fluorescence.

PubMed

Rawel, Harshadrai M; Frey, Simone K; Meidtner, Karina; Kroll, Jürgen; Schweigert, Florian J

2006-08-01

The noncovalent binding of selected phenolic compounds (chlorogenic-, ferulic-, gallic acid, quercetin, rutin, and isoquercetin) to proteins (HSA, BSA, soy glycinin, and lysozyme) was studied by an indirect method applying the quenching of intrinsic tryptophan fluorescence. From the data obtained, the binding constants were calculated by nonlinear regression (one site binding; y = Bx/k + x). It has been reported that tannins inhibit human salivary amylase and that these complexes may reduce the development of cariogenic plaques. Further, amylase contains two tryptophan residues in its active site. Therefore, in a second part of the study involving 31 human subjects, evidence was sought for noncovalent interactions between the phenols of green tea and saliva proteins as measured by the fluorescence intensity. Amylase activity was determined before and after the addition of green tea to saliva of 31 subjects. Forty percent of the subjects showed an increase in amylase activity contrary to studies reporting only a decrease in activity. The interactions of tannin with amylase result in a decrease of its activity. It still remains to be elucidated why amylase does not react uniformly under conditions of applying green tea to saliva. Further, in terms of using phenols as caries inhibitors this finding should be of importance.

Protein sorption on polymer surfaces measured by fluorescence labels.

PubMed

Brynda, E; Drobník, J; Vacík, J; Kálal, J

1978-01-01

Fluorescence labeling can be used in studying protein sorption on various surfaces with a sensitivity of about 10(-8) g/cm2, commensurate with radioactive labeling. Fluorescamine proved to be the most suitable compound for studying protein sorption on hydrophilic gels, because, unlike fluoresceine isothiocyanate and dansylchloride, free fluorochrome does not interfere with measurements. Sorption properties of labeled serum albumin were tested on poly(2-hydroxyethyl methacrylate), on the copolymer of 2-hydroxyethyl methacrylate with methyl methacrylate, and on polyethylene. Labeling does not cause aggregation of the protein, but, as expected, it shifts and somewhat broadens its electrophoretic band while at the same time slightly raising its affinity toward hydrophobic surfaces.

Targeting protein-protein interactions with trimeric ligands: high affinity inhibitors of the MAGUK protein family.

PubMed

Nissen, Klaus B; Haugaard-Kedström, Linda M; Wilbek, Theis S; Nielsen, Line S; Åberg, Emma; Kristensen, Anders S; Bach, Anders; Jemth, Per; Strømgaard, Kristian

2015-01-01

PDZ domains in general, and those of PSD-95 in particular, are emerging as promising drug targets for diseases such as ischemic stroke. We have previously shown that dimeric ligands that simultaneously target PDZ1 and PDZ2 of PSD-95 are highly potent inhibitors of PSD-95. However, PSD-95 and the related MAGUK proteins contain three consecutive PDZ domains, hence we envisioned that targeting all three PDZ domains simultaneously would lead to more potent and potentially more specific interactions with the MAGUK proteins. Here we describe the design, synthesis and characterization of a series of trimeric ligands targeting all three PDZ domains of PSD-95 and the related MAGUK proteins, PSD-93, SAP-97 and SAP-102. Using our dimeric ligands targeting the PDZ1-2 tandem as starting point, we designed novel trimeric ligands by introducing a PDZ3-binding peptide moiety via a cysteine-derivatized NPEG linker. The trimeric ligands generally displayed increased affinities compared to the dimeric ligands in fluorescence polarization binding experiments and optimized trimeric ligands showed low nanomolar inhibition towards the four MAGUK proteins, thus being the most potent inhibitors described. Kinetic experiments using stopped-flow spectrometry showed that the increase in affinity is caused by a decrease in the dissociation rate of the trimeric ligand as compared to the dimeric ligands, likely reflecting the lower probability of simultaneous dissociation of all three PDZ ligands. Thus, we have provided novel inhibitors of the MAGUK proteins with exceptionally high affinity, which can be used to further elucidate the therapeutic potential of these proteins.

Dual-Modal Magnetic Resonance/Fluorescent Zinc Probes for Pancreatic β-Cell Mass Imaging

PubMed Central

Stasiuk, Graeme J; Minuzzi, Florencia; Sae-Heng, Myra; Rivas, Charlotte; Juretschke, Hans-Paul; Piemonti, Lorenzo; Allegrini, Peter R; Laurent, Didier; Duckworth, Andrew R; Beeby, Andrew; Rutter, Guy A; Long, Nicholas J

2015-01-01

Despite the contribution of changes in pancreatic β-cell mass to the development of all forms of diabetes mellitus, few robust approaches currently exist to monitor these changes prospectively in vivo. Although magnetic-resonance imaging (MRI) provides a potentially useful technique, targeting MRI-active probes to the β cell has proved challenging. Zinc ions are highly concentrated in the secretory granule, but they are relatively less abundant in the exocrine pancreas and in other tissues. We have therefore developed functional dual-modal probes based on transition-metal chelates capable of binding zinc. The first of these, Gd⋅1, binds ZnII directly by means of an amidoquinoline moiety (AQA), thus causing a large ratiometric Stokes shift in the fluorescence from λem=410 to 500 nm with an increase in relaxivity from r1=4.2 up to 4.9 mM−1 s−1. The probe is efficiently accumulated into secretory granules in β-cell-derived lines and isolated islets, but more poorly by non-endocrine cells, and leads to a reduction in T1 in human islets. In vivo murine studies of Gd⋅1 have shown accumulation of the probe in the pancreas with increased signal intensity over 140 minutes. PMID:25736590

Interferences of Silica Nanoparticles in Green Fluorescent Protein Folding Processes.

PubMed

Klein, Géraldine; Devineau, Stéphanie; Aude, Jean Christophe; Boulard, Yves; Pasquier, Hélène; Labarre, Jean; Pin, Serge; Renault, Jean Philippe

2016-01-12

We investigated the relationship between unfolded proteins, silica nanoparticles and chaperonin to determine whether unfolded proteins could stick to silica surfaces and how this process could impair heat shock protein activity. The HSP60 catalyzed green fluorescent protein (GFP) folding was used as a model system. The adsorption isotherms and adsorption kinetics of denatured GFP were measured, showing that denaturation increases GFP affinity for silica surfaces. This affinity is maintained even if the surfaces are covered by a protein corona and allows silica NPs to interfere directly with GFP folding by trapping it in its unstructured state. We determined also the adsorption isotherms of HSP60 and its chaperonin activity once adsorbed, showing that SiO2 NP can interfere also indirectly with protein folding through chaperonin trapping and inhibition. This inhibition is specifically efficient when NPs are covered first with a layer of unfolded proteins. These results highlight for the first time the antichaperonin activity of silica NPs and ask new questions about the toxicity of such misfolded proteins/nanoparticles assembly toward cells.

The alpha3(betaMet222Ser/Tyr345Trp)3gamma subcomplex of the TF1-ATPase does not hydolyze ATP at a significant rate until the substrate binds to the catalytic site of the lowest affinity.

PubMed

Ren, Huimiao; Bandyopadhyay, Sanjay; Allison, William S

2006-05-16

The alpha(3)(betaM(222)S/Y(345)W)(3)gamma double-mutant subcomplex of the F(1)-ATPase from the thermophilic Bacillus PS3 (TF(1)), free of endogenous nucleotides, does not entrap inhibitory MgADP in a catalytic site during turnover. It hydrolyzes 100 nM-2 mM ATP with a K(m) of 31 microM and a k(cat) of 220 s(-)(1). Fluorescence titrations of the introduced tryptophans with MgADP or MgATP revealed that both Mg-nucleotide complexes bind to the catalytic site of the highest affinity with K(d)()1 values of less than 1 nM and bind to the site of intermediate affinity with a common K(d)2 value of about 12 nM. The K(d)3 values obtained for the catalytic site of the lowest affinity from titrations with MgADP and MgATP are 25 and 37 microM, respectively. The double mutant hydrolyzes 200 nM ATP with a first-order rate of 1.5 s(-)(1), which is 0.7% of k(cat). Hence, it does not hydrolyze ATP at a significant rate when the catalytic site of intermediate affinity is saturated and the catalytic site of the lowest affinity is minimally occupied. After the addition of stoichiometric MgATP to the alpha(3)(betaM(222)S/Y(345)W)(3)gamma subcomplex, one-third of the tryptophan fluorescence remains quenched after 10 min. The product [(3)H]ADP remains bound when the wild-type and double-mutant subcomplexes hydrolyze substoichiometric [(3)H]ATP. In contrast, (32)P(i) is not retained when the wild-type subcomplex hydrolyzes substoichiometric [gamma-(32)P]ATP. This precludes assessment of the equilibrium at the high-affinity catalytic site when the wild-type TF(1) subcomplex hydrolyzes substoichiometric ATP.

Notch and affinity boundaries in Drosophila.

PubMed

Herranz, Héctor; Milán, Marco

2006-02-01

Cells in multicellular organisms often do not intermingle freely with each other. Differential cell affinities can contribute to organizing cells into different tissues. Drosophila limbs and the vertebrate central nervous system are subdivided into compartments. Cells in adjacent compartments do not mix. Cell interactions mediated by Notch-family receptors have been implicated in the specification of these compartment boundaries. Two recent reports analyze the role of the Notch signaling pathway in the generation of an affinity boundary in the Drosophila wing. The first report analyzes the connection between Notch and the actin cytoskeleton. The second report analyzes the differential requirements of Notch and the transcription factor Suppressor of Hairless in generating the affinity boundary.

Investigation on the pH-dependent binding of benzocaine and lysozyme by fluorescence and absorbance

NASA Astrophysics Data System (ADS)

Li, Shihui; Li, Daojin

2011-11-01

The interaction mechanism between benzocaine (BZC) and lysozyme (Lys) has been investigated by fluorescence, synchronous fluorescence, ultraviolet-vis (UV) absorption spectra, and three-dimensional fluorescence (3-D) in various pH medium. The observations of fluorescence spectra were mainly rationalized in terms of a static quenching process at lower concentration of BZC ( CBZC/ CLys < 9) and a combined quenching process at higher concentration of BZC ( CBZC/ CLys > 9) at pH 7.4 and 8.4. However, the fluorescence quenching was mainly arisen from static quenching by complex formation in all studied drug concentrations at pH 3.5. The structural characteristics of BZC and Lys were probed, and their binding affinities were determined under different pH conditions (pH 3.5, 7.4, and 8.4). The results indicated that the binding abilities of BZC to Lys decreased at the pH below and above the simulative physiological condition (pH 7.4) due to the alterations of the protein secondary and tertiary structures or the structural change of BZC. The effect of BZC on the conformation of Lys was analyzed using UV, synchronous fluorescence and three-dimensional fluorescence under different pH conditions. These results indicate that the binding of BZC to Lys causes apparent change in the secondary and tertiary structures of Lys. The effect of Zn 2+ on the binding constant of BZC with Lys under various pH conditions (pH 3.5, 7.4, and 8.4) was also studied.

A cooperative-binding split aptamer assay for rapid, specific and ultra-sensitive fluorescence detection of cocaine in saliva.

PubMed

Yu, Haixiang; Canoura, Juan; Guntupalli, Bhargav; Lou, Xinhui; Xiao, Yi

2017-01-01

Sensors employing split aptamers that reassemble in the presence of a target can achieve excellent specificity, but the accompanying reduction of target affinity mitigates any overall gains in sensitivity. We for the first time have developed a split aptamer that achieves enhanced target-binding affinity through cooperative binding. We have generated a split cocaine-binding aptamer that incorporates two binding domains, such that target binding at one domain greatly increases the affinity of the second domain. We experimentally demonstrate that the resulting cooperative-binding split aptamer (CBSA) exhibits higher target binding affinity and is far more responsive in terms of target-induced aptamer assembly compared to the single-domain parent split aptamer (PSA) from which it was derived. We further confirm that the target-binding affinity of our CBSA can be affected by the cooperativity of its binding domains and the intrinsic affinity of its PSA. To the best of our knowledge, CBSA-5335 has the highest cocaine affinity of any split aptamer described to date. The CBSA-based assay also demonstrates excellent performance in target detection in complex samples. Using this CBSA, we achieved specific, ultra-sensitive, one-step fluorescence detection of cocaine within fifteen minutes at concentrations as low as 50 nM in 10% saliva without signal amplification. This limit of detection meets the standards recommended by the European Union's Driving under the Influence of Drugs, Alcohol and Medicines program. Our assay also demonstrates excellent reproducibility of results, confirming that this CBSA-platform represents a robust and sensitive means for cocaine detection in actual clinical samples.

Application of Effective Fragment Potential Methos to the Redox Potential of Green Fluorescent Protein

NASA Astrophysics Data System (ADS)

Ghosh, Debashree; Krylov, Anna I.

2011-06-01

Green fluorescent proteins (GFP) can be considered as a model for flurogenic dyes and are of importance in photovoltaic materials. It exhibits bright green fluorescence when exposed to blue light and has been an extremely powerful tool as non-invasive marker in living cells and extensibly used in molecular and cell biology. The understanding of the underlying electronic structure of these proteins and its chromophore is therefore crucial to the understanding of the mechanism for its optical properties. The chromophore of the GFP is p-hydroxybenzylidene-imidazolinone (HBDI) and is embedded in the center of the β barrel of the GFP. Calculating redox potential of this chromophore is a challenging problem, especially in diverse solvents and protein environment. It is possible to carry out high-level accurate ab-initio calculation of ionization potential or electron affinity of the microsolvated chromophore or the bare chromophore. But, it is not possible to extend these calculations to bulk solvents due to the high computational cost. Effective fragment potential (EFP)[1,2] method gives us a convenient tool to understand such systems. In our work, we have benchmarked the ionization energy and electron affinity of the microsolvated GFP chromophore calculated by combined EOM-IP-CCSD/EFP and EOM-EA-CCSD/EFP with the EOM-IP-CCSD and EOM-EA-CCSD calculations of the oxidized and reduced forms. We have carried out similar EFP-EOM-IP-CCSD and EFP-EOM-EA-CCSD calculations of ionization potential and electron affinity of GFP choromophore in bulk solvent generated by ab-initio molecular dynamics simulations. [1] M. S. Gordon, L. Slipchenko, H. Li, J. H. Jensen, Annual Reports in Computational Chemistry, Volume 3, 177 (2007). [2] D. Ghosh, D. Kosenkov, V. Vanovschi, C.F. Williams, J.M. Herbert, M.S. Gordon, M.W. Schmidt, L.V. Slipchenko, and A.I. Krylov, J. Phys. Chem. A 114, 12739 (2010).

PHARMACEUTICAL AND BIOMEDICAL APPLICATIONS OF AFFINITY CHROMATOGRAPHY: RECENT TRENDS AND DEVELOPMENTS

PubMed Central

Hage, David S.; Anguizola, Jeanethe A.; Bi, Cong; Li, Rong; Matsuda, Ryan; Papastavros, Efthimia; Pfaunmiller, Erika; Vargas, John; Zheng, Xiwei

2012-01-01

Affinity chromatography is a separation technique that has become increasingly important in work with biological samples and pharmaceutical agents. This method is based on the use of a biologically-related agent as a stationary phase to selectively retain analytes or to study biological interactions. This review discusses the basic principles behind affinity chromatography and examines recent developments that have occurred in the use of this method for biomedical and pharmaceutical analysis. Techniques based on traditional affinity supports are discussed, but an emphasis is placed on methods in which affinity columns are used as part of HPLC systems or in combination with other analytical methods. General formats for affinity chromatography that are considered include step elution schemes, weak affinity chromatography, affinity extraction and affinity depletion. Specific separation techniques that are examined include lectin affinity chromatography, boronate affinity chromatography, immunoaffinity chromatography, and immobilized metal ion affinity chromatography. Approaches for the study of biological interactions by affinity chromatography are also presented, such as the measurement of equilibrium constants, rate constants, or competition and displacement effects. In addition, related developments in the use of immobilized enzyme reactors, molecularly imprinted polymers, dye ligands and aptamers are briefly considered. PMID:22305083

High-affinity K+ uptake in pepper plants.

PubMed

Martínez-Cordero, M Angeles; Martínez, Vicente; Rubio, Francisco

2005-06-01

High-affinity K+ uptake is an essential process for plant nutrition under K+-limiting conditions. The results presented here demonstrate that pepper (Capsicum annuum) plants grown in the absence of NH4+ and starved of K+ show an NH4+-sensitive high-affinity K+ uptake that allows plant roots to deplete external K+ to values below 1 microM. When plants are grown in the presence of NH4+, high-affinity K+ uptake is not inhibited by NH4+. Although NH4+-grown plants deplete external K+ below 1 microM in the absence of NH4+, when 1 mM NH4+ is present they do not deplete external K+ below 10 microM. A K+ transporter of the HAK family, CaHAK1, is very likely mediating the NH4+-sensitive component of the high-affinity K+ uptake in pepper roots. CaHAK1 is strongly induced in the roots that show the NH4+-sensitive high-affinity K+ uptake and its induction is reduced in K+-starved plants grown in the presence of NH4+. The NH4+-insensitive K+ uptake may be mediated by an AKT1-like K+ channel.

Molecular Structure-Affinity Relationship of Flavonoids in Lotus Leaf (Nelumbo nucifera Gaertn.) on Binding to Human Serum Albumin and Bovine Serum Albumin by Spectroscopic Method.

PubMed

Tang, Xiaosheng; Tang, Ping; Liu, Liangliang

2017-06-23

Lotus leaf has gained growing popularity as an ingredient in herbal formulations due to its various activities. As main functional components of lotus leaf, the difference in structure of flavonoids affected their binding properties and activities. In this paper, the existence of 11 flavonoids in lotus leaf extract was confirmed by High Performance Liquid Chromatography (HPLC) analysis and 11 flavonoids showed various contents in lotus leaf. The interactions between lotus leaf extract and two kinds of serum albumins (human serum albumin (HSA) and bovine serum albumin (BSA)) were investigated by spectroscopic methods. Based on the fluorescence quenching, the interactions between these flavonoids and serum albumins were further checked in detail. The relationship between the molecular properties of flavonoids and their affinities for serum albumins were analyzed and compared. The hydroxylation on 3 and 3' position increased the affinities for serum albumins. Moreover, both of the methylation on 3' position of quercetin and the C₂=C₃ double bond of apigenin and quercetin decreased the affinities for HSA and BSA. The glycosylation lowered the affinities for HSA and BSA depending on the type of sugar moiety. It revealed that the hydrogen bond force played an important role in binding flavonoids to HSA and BSA.

Electron affinity of liquid water

DOE PAGES

Gaiduk, Alex P.; Pham, Tuan Anh; Govoni, Marco; ...

2018-01-16

Understanding redox and photochemical reactions in aqueous environments requires a precise knowledge of the ionization potential and electron affinity of liquid water. The former has been measured, but not the latter. We predict the electron affinity of liquid water and of its surface from first principles, coupling path-integral molecular dynamics with ab initio potentials, and many-body perturbation theory. Our results for the surface (0.8 eV) agree well with recent pump-probe spectroscopy measurements on amorphous ice. Those for the bulk (0.1-0.3 eV) differ from several estimates adopted in the literature, which we critically revisit. We show that the ionization potential ofmore » the bulk and surface are almost identical; instead their electron affinities differ substantially, with the conduction band edge of the surface much deeper in energy than that of the bulk. We also discuss the significant impact of nuclear quantum effects on the fundamental gap and band edges of the liquid.« less

Electron affinity of liquid water

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gaiduk, Alex P.; Pham, Tuan Anh; Govoni, Marco

Understanding redox and photochemical reactions in aqueous environments requires a precise knowledge of the ionization potential and electron affinity of liquid water. The former has been measured, but not the latter. We predict the electron affinity of liquid water and of its surface from first principles, coupling path-integral molecular dynamics with ab initio potentials, and many-body perturbation theory. Our results for the surface (0.8 eV) agree well with recent pump-probe spectroscopy measurements on amorphous ice. Those for the bulk (0.1-0.3 eV) differ from several estimates adopted in the literature, which we critically revisit. We show that the ionization potential ofmore » the bulk and surface are almost identical; instead their electron affinities differ substantially, with the conduction band edge of the surface much deeper in energy than that of the bulk. We also discuss the significant impact of nuclear quantum effects on the fundamental gap and band edges of the liquid.« less

Stopped-flow studies of changes in fluorescence of 8-anilino-1-naphthalene sulfonic acid caused by magnesium and salt binding to yeast enolase.

PubMed

Brewer, J M

1976-12-11

Stopped-flow studies of magnesium and salt (potassium chloride and acetate) effects on yeast enolase were carried out by following 8-anilino-1-naphthalenesulfonic acid fluorescence changes. The fluorescence changes appear to be largely caused by subunit association and dissociation, though there is evidence in some reactions for large changes in fluorescence occurring within the dead time of the stopped-flow measurements. These data are combined with measurements of initial enzyme activity after incubation in various solvents with or without magnesium to obtain subunit association and dissociation rates. From these, it is concluded that magnesium and the salts act by directly changing the affinities of the subunits for each other, apparently by producing a rapid change in protein conformation.

Removal of Pb(II), Cd(II), Cu(II), and Zn(II) by hematite nanoparticles: effect of sorbent concentration, pH, temperature, and exhaustion.

PubMed

Shipley, Heather J; Engates, Karen E; Grover, Valerie A

2013-03-01

Nanoparticles offer the potential to improve environmental treatment technologies due to their unique properties. Adsorption of metal ions (Pb(II), Cd(II), Cu(II), Zn(II)) to nanohematite was examined as a function of sorbent concentration, pH, temperature, and exhaustion. Adsorption experiments were conducted with 0.05, 0.1, and 0.5 g/L nanoparticles in a pH 8 solution and in spiked San Antonio tap water. The adsorption data showed the ability of nanohematite to remove Pb, Cd, Cu, and Zn species from solution with adsorption increasing as the nanoparticle concentration increased. At 0.5 g/L nanohematite, 100 % Pb species adsorbed, 94 % Cd species adsorbed, 89 % Cu species adsorbed and 100 % Zn species adsorbed. Adsorption kinetics for all metals tested was described by a pseudo second-order rate equation with lead having the fastest rate of adsorption. The effect of temperature on adsorption showed that Pb(II), Cu(II), and Cd(II) underwent an endothermic reaction, while Zn(II) underwent an exothermic reaction. The nanoparticles were able to simultaneously remove multiple metals species (Zn, Cd, Pb, and Cu) from both a pH 8 solution and spiked San Antonio tap water. Exhaustion experiments showed that at pH 8, exhaustion did not occur for the nanoparticles but adsorption does decrease for Cd, Cu, and Zn species but not Pb species. The strong adsorption coupled with the ability to simultaneously remove multiple metal ions offers a potential remediation method for the removal of metals from water.

Phenoxo bridged dinuclear Zn(II) Schiff base complex as new precursor for preparation zinc oxide nanoparticles: Synthesis, characterization, crystal structures and photoluminescence studies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saeednia, S., E-mail: sami_saeednia@yahoo.com; Iranmanesh, P.; Ardakani, M. Hatefi

Highlights: • A novel nano-scale Zn(II) complex was synthesized by solvothermal method. • Chemical structure of the nanostructures was characterized as well as bulk complex. • The photoluminescence property of the complex was investigated at room temperature. • The thermogravimetry and differential thermal analysis were carried out. • Thermal decomposition of the nanostructures was prepared zinc oxide nanoparticles. - Abstract: Nanoparticles of a novel Zn(II) Schiff base complex, [Zn(HL)NO{sub 3}]{sub 2} (1), (H{sub 2}L = 2-[(2-hydroxy-propylimino) methyl] phenol), was synthesized by using solvothermal method. Shape, morphology and chemical structure of the synthesized nanoparticles were characterized by scanning electron microscopy (SEM),more » X-ray powder diffraction (XRD), Fourier Transform Infrared Spectoscopy (FT-IR) and UV–vis spectroscopy. Structural determination of compound 1 was determined by single-crystal X-ray diffraction. The results were revealed that the zinc complex is a centrosymmetric dimer in which deprotonated phenolates bridge the two five-coordinate metal atoms and link the two halves of the dimer. The thermal stability of compound 1 was analyzed by thermal gravimetric analysis (TGA) and differential scanning calorimetry (DSC). The effect of the initial substrates concentration and reaction time on size and morphology of compound 1 nanostructure was investigated as well. Furthermore, the luminescent properties of the complex 1 were examined. ZnO nanoparticles with diameter between 15 and 20 nm were simply synthesized by solid-state transformation of compound 1 at 700 °C.« less

How Structure Defines Affinity in Protein-Protein Interactions

PubMed Central

Erijman, Ariel; Rosenthal, Eran; Shifman, Julia M.

2014-01-01

Protein-protein interactions (PPI) in nature are conveyed by a multitude of binding modes involving various surfaces, secondary structure elements and intermolecular interactions. This diversity results in PPI binding affinities that span more than nine orders of magnitude. Several early studies attempted to correlate PPI binding affinities to various structure-derived features with limited success. The growing number of high-resolution structures, the appearance of more precise methods for measuring binding affinities and the development of new computational algorithms enable more thorough investigations in this direction. Here, we use a large dataset of PPI structures with the documented binding affinities to calculate a number of structure-based features that could potentially define binding energetics. We explore how well each calculated biophysical feature alone correlates with binding affinity and determine the features that could be used to distinguish between high-, medium- and low- affinity PPIs. Furthermore, we test how various combinations of features could be applied to predict binding affinity and observe a slow improvement in correlation as more features are incorporated into the equation. In addition, we observe a considerable improvement in predictions if we exclude from our analysis low-resolution and NMR structures, revealing the importance of capturing exact intermolecular interactions in our calculations. Our analysis should facilitate prediction of new interactions on the genome scale, better characterization of signaling networks and design of novel binding partners for various target proteins. PMID:25329579

Affinity and specificity of interactions between Nedd4 isoforms and the epithelial Na+ channel.

PubMed

Henry, Pauline C; Kanelis, Voula; O'Brien, M Christine; Kim, Brian; Gautschi, Ivan; Forman-Kay, Julie; Schild, Laurent; Rotin, Daniela

2003-05-30

The epithelial Na+ channel (alphabetagammaENaC) regulates salt and fluid homeostasis and blood pressure. Each ENaC subunit contains a PY motif (PPXY) that binds to the WW domains of Nedd4, a Hect family ubiquitin ligase containing 3-4 WW domains and usually a C2 domain. It has been proposed that Nedd4-2, but not Nedd4-1, isoforms can bind to and suppress ENaC activity. Here we challenge this notion and show that, instead, the presence of a unique WW domain (WW3*) in either Nedd4-2 or Nedd4-1 determines high affinity interactions and the ability to suppress ENaC. WW3* from either Nedd4-2 or Nedd4-1 binds ENaC-PY motifs equally well (e.g. Kd approximately 10 microm for alpha- or betaENaC, 3-6-fold higher affinity than WW4), as determined by intrinsic tryptophan fluorescence. Moreover, dNedd4-1, which naturally contains a WW3* instead of WW2, is able to suppress ENaC function equally well as Nedd4-2. Homology models of the WW3*.betaENaC-PY complex revealed that a Pro and Ala conserved in all WW3*, but not other Nedd4-WW domains, help form the binding pocket for PY motif prolines. Extensive contacts are formed between the betaENaC-PY motif and the Pro in WW3*, and the small Ala creates a large pocket to accommodate the peptide. Indeed, mutating the conserved Pro and Ala in WW3* reduces binding affinity 2-3-fold. Additionally, we demonstrate that mutations in PY motif residues that form contacts with the WW domain based on our previously solved structure either abolish or severely reduce binding affinity to the WW domain and that the extent of binding correlates with the level of ENaC suppression. Independently, we show that a peptide encompassing the PY motif of sgk1, previously proposed to bind to Nedd4-2 and alter its ability to regulate ENaC, does not bind (or binds poorly) the WW domains of Nedd4-2. Collectively, these results suggest that high affinity of WW domain-PY-motif interactions rather than affiliation with Nedd4-1/Nedd-2 is critical for ENaC suppression

Development of Substrate-Selective Probes for Affinity Pulldown of Histone Demethylases

PubMed Central

2015-01-01

JmjC-domain containing histone demethylases (JHDMs) play critical roles in many key cellular processes and have been implicated in multiple disease conditions. Each enzyme within this family is known to have a strict substrate scope, specifically the position of the lysine within the histone and its degree of methylation. While much progress has been made in determining the substrates of each enzyme, new methods with which to systematically profile each histone mark are greatly needed. Novel chemical tools have the potential to fill this role and, furthermore, can be used as probes to answer fundamental questions about these enzymes and serve as potential therapeutic leads. In this work, we first investigated three small-molecule probes differing in the degree of “methylation state” and their differential bindings to JHDM1A (an H3K36me1/2 demethylase) using a fluorescence polarization-based competition assay. We then applied this specificity toward the “methylation state” and combined it with specificity toward lysine position in the design and synthesis of a peptidic probe targeting H3K36me2 JHDMs. The probe is further functionalized with a benzophenone cross-linking moiety and a biotin for affinity purification. Results showed binding of the peptidic probe to JHDM1A and specific enrichment of this protein in the presence of its native histone substrates. Affinity purification pulldown experiments from nuclear lysate coupled with mass spectrometry revealed the capability of the probe to pull out and enrich JHDMs along with other epigenetic proteins and transcriptional regulators. PMID:25335116

Synthesis, structure, luminescent, and magnetic properties of carbonato-bridged Zn(II)2Ln(III)2 complexes [(μ4-CO3)2{Zn(II)L(n)Ln(III)(NO3)}2] (Ln(III) = Gd(III), Tb(III), Dy(III); L(1) = N,N'-bis(3-methoxy-2-oxybenzylidene)-1,3-propanediaminato, L(2) = N,N'-bis(3-ethoxy-2-oxybenzylidene)-1,3-propanediaminato).

PubMed

Ehama, Kiyomi; Ohmichi, Yusuke; Sakamoto, Soichiro; Fujinami, Takeshi; Matsumoto, Naohide; Mochida, Naotaka; Ishida, Takayuki; Sunatsuki, Yukinari; Tsuchimoto, Masanobu; Re, Nazzareno

2013-11-04

Carbonato-bridged Zn(II)2Ln(III)2 complexes [(μ4-CO3)2{Zn(II)L(n)Ln(III)(NO3)}2]·solvent were synthesized through atmospheric CO2 fixation reaction of [Zn(II)L(n)(H2O)2]·xH2O, Ln(III)(NO3)3·6H2O, and triethylamine, where Ln(III) = Gd(III), Tb(III), Dy(III); L(1) = N,N'-bis(3-methoxy-2-oxybenzylidene)-1,3-propanediaminato, L(2) = N,N'-bis(3-ethoxy-2-oxybenzylidene)-1,3-propanediaminato. Each Zn(II)2Ln(III)2 structure possessing an inversion center can be described as two di-μ-phenoxo-bridged {Zn(II)L(n)Ln(III)(NO3)} binuclear units bridged by two carbonato CO3(2-) ions. The Zn(II) ion has square pyramidal coordination geometry with N2O2 donor atoms of L(n) and one oxygen atom of a bridging carbonato ion at the axial site. Ln(III) ion is coordinated by nine oxygen atoms consisting of four from the deprotonated Schiff-base L(n), two from a chelating nitrate, and three from two carbonate groups. The temperature-dependent magnetic susceptibilities in the range 1.9-300 K, field-dependent magnetization from 0 to 5 T at 1.9 K, and alternating current magnetic susceptibilities under the direct current bias fields of 0 and 1000 Oe were measured. The magnetic properties of the Zn(II)2Ln(III)2 complexes are analyzed on the basis of the dicarbonato-bridged binuclear Ln(III)-Ln(III) structure, as the Zn(II) ion with d(10) electronic configuration is diamagnetic. ZnGd1 (L(1)) and ZnGd2 (L(2)) show a ferromagnetic Gd(III)-Gd(III) interaction with J(Gd-Gd) = +0.042 and +0.028 cm(-1), respectively, on the basis of the Hamiltonian H = -2J(Gd-Gd)ŜGd1·ŜGd2. The magnetic data of the Zn(II)2Ln(III)2 complexes (Ln(III) = Tb(III), Dy(III)) were analyzed by a spin Hamiltonian including the crystal field effect on the Ln(III) ions and the Ln(III)-Ln(III) magnetic interaction. The Stark splitting of the ground state was so evaluated, and the energy pattern indicates a strong easy axis (Ising type) anisotropy. Luminescence spectra of Zn(II)2Tb(III)2 complexes were observed, while those

Synthesis, crystal structures, spectral, thermal and antimicrobial properties of new Zn(II) 5-iodo- and 5-bromosalicylates

NASA Astrophysics Data System (ADS)

Košická, Petra; Győryová, Katarína; Smolko, Lukáš; Gyepes, Róbert; Hudecová, Daniela

2018-03-01

Two new analogous zinc(II) complexes containing 5-iodo- and 5-bromosalicylate ligands, respectively, were prepared in single-crystal form and characterized by IR spectroscopy, thermal analysis and elemental analysis. The solid-state structures of prepared complexes were determined by single crystal X-ray crystallography. Both complexes are isostructural and their crystal structures composed of neutral molecules [Zn(5-Xsal)2(H2O)2] (where X = Br, I, sal = salicylato). Central Zn(II) atom is in both complexes coordinated by six oxygen atoms, four of which are from two chelate bonded 5-halosalicylates and remaining two from coordinated water molecules. The found chelate binding mode is in line with the Δ values calculated from IR spectral data. Antimicrobial activity of prepared complexes was studied against selected bacteria, yeast and filamentous fungi. Obtained results indicate that 5-iodosalicylate complex is more antimicrobially active than its 5-bromo substituted analogue.

Combined EXAFS and DFT Structure Calculations Provide Structural Insights into the 1:1 Multi-Histidine Complexes of CuII, CuI and ZnII with the Tandem Octarepeats of the Mammalian Prion Protein

PubMed Central

Pushie, M. Jake; Nienaber, Kurt H.; McDonald, Alex; Millhauser, Glenn L.; George, Graham N.

2014-01-01

The metal coordinating properties of the prion protein (PrP) have been the subject of intense focus and debate since the first reports of copper interaction with PrP just before the turn of the century. The picture of metal coordination to PrP has been improved and refined over the past decade, and yet the structural details of the various metal coordination modes have not been fully elucidated in some cases. Herein we employ X-ray absorption near edge spectroscopy as well as extended X-ray absorption fine structure (EXAFS) spectroscopy to structurally characterize the dominant 1:1 coordination modes for CuII, CuI and ZnII with an N-terminal fragment of PrP. The PrP fragment constitutes four tandem repeats representative of the mammalian octarepeat domain, designated OR4, which is also the most studied PrP fragment for metal interactions, making our findings applicable to a large body of previous work. Density functional theory (DFT) calculations provide additional structural and thermodynamic data, and candidate structures are used to inform EXAFS data analysis. The optimized geometries from DFT calculations are used to identify potential coordination complexes for multi-histidine coordination of CuII, CuI and ZnII in an aqueous medium, modeled using 4-methylimidazole to represent the histidine side chain. Through a combination of in silico coordination chemistry as well as rigorous EXAFS curve fitting, using full multiple scattering on candidate structures from DFT calculations, we have characterized the predominant coordination modes for the 1:1 complexes of CuII, CuI and ZnII with the OR4 peptide at pH 7.4 at atomic resolution, which are best represented as a square planar [CuII(His)4]2+, digonal [CuI(His)2]+ and tetrahedral [ZnII(His)3(OH2)]2+, respectively. PMID:25042361

A supramolecular Tröger's base derived coordination zinc polymer for fluorescent sensing of phenolic-nitroaromatic explosives in water† †Electronic supplementary information (ESI) available: Spectroscopic (multinuclear NMR, IR HRMS) characterizations, optimized structure, TGA, powder diffraction and fluorescence titration profiles. See DOI: 10.1039/c6sc04367d Click here for additional data file.

PubMed Central

Dabadie, Charlyne; Byrne, Kevin; Savyasachi, Aramballi J.; Umadevi, Deivasigamani; Schmitt, Wolfgang; Kitchen, Jonathan A.

2017-01-01

A V-Shaped 4-amino-1,8-napthalimide derived tetracarboxylic acid linker (L; bis-[N-(1,3-benzenedicarboxylic acid)]-9,18-methano-1,8-naphthalimide-[b,f][1,5]diazocine) comprising the Tröger's base (TB) structural motif was rationally designed and synthesised to access a nitrogen-rich fluorescent supramolecular coordination polymer. By adopting the straight forward precipitation method, a new luminescent nanoscale Zn(ii) coordination polymer (TB-Zn-CP) was synthesized in quantitative yield using Zn(OAc)2·2H2O and tetraacid linker L (1 : 0.5) in DMF at room temperature. The phase-purity of as-synthesised TB-Zn-CP was confirmed by X-ray powder diffraction analysis, infra-red spectroscopy, and elemental analysis. Thermogravimetric analysis suggests that TB-Zn-CP is thermally stable up to 330 °C and the morphological features of TB-Zn-CP was analysed by SEM and AFM techniques. The N2 adsorption isotherm of thermally activated TB-Zn-CP at 77 K revealed a type-II reversible adsorption isotherm and the calculated Brunauer–Emmett–Teller (BET) surface area was found to be 72 m2 g–1. Furthermore, TB-Zn-CP displayed an excellent CO2 uptake capacity of 76 mg g–1 at 273 K and good adsorption selectivity for CO2 over N2 and H2. The aqueous suspension of as-synthesized TB-Zn-CP showed strong green fluorescence (λ max = 520 nm) characteristics due to the internal-charge transfer (ICT) transition and was used as a fluorescent sensor for the discriminative sensing of nitroaromatic explosives. The aqueous suspension of TB-Zn-CP showed the largest quenching responses with high selectivity for phenolic-nitroaromatics (4-NP, 2,4-DNP and PA) even in the concurrent presence of other potentially competing nitroaromatic analytes. The fluorescence titration studies also provide evidence that TB-Zn-CP detects picric acid as low as the parts per billion (26.3 ppb) range. Furthermore, the observed fluorescence quenching responses of TB-Zn-CP towards picric acid were highly

Employees' Perceptions of Female Affinity Groups

ERIC Educational Resources Information Center

Gabriel, Peter J.

2017-01-01

Female affinity groups offered by corporations are formalized corporate initiatives focused on networking and professional development opportunities for women (Welbourne & McLaughlin, 2013). The scholarly literature focused on female affinity groups was limited. The purpose of this grounded theory Dissertation in Practice study was to…

Affinity+: Semi-Structured Brainstorming on Large Displays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Burtner, Edwin R.; May, Richard A.; Scarberry, Randall E.

2013-04-27

Affinity diagraming is a powerful method for encouraging and capturing lateral thinking in a group environment. The Affinity+ Concept was designed to improve the collaborative brainstorm process through the use of large display surfaces in conjunction with mobile devices like smart phones and tablets. The system works by capturing the ideas digitally and allowing users to sort and group them on a large touch screen manually. Additionally, Affinity+ incorporates theme detection, topic clustering, and other processing algorithms that help bring structured analytic techniques to the process without requiring explicit leadership roles and other overhead typically involved in these activities.

Classification of neocortical interneurons using affinity propagation.

PubMed

Santana, Roberto; McGarry, Laura M; Bielza, Concha; Larrañaga, Pedro; Yuste, Rafael

2013-01-01

In spite of over a century of research on cortical circuits, it is still unknown how many classes of cortical neurons exist. In fact, neuronal classification is a difficult problem because it is unclear how to designate a neuronal cell class and what are the best characteristics to define them. Recently, unsupervised classifications using cluster analysis based on morphological, physiological, or molecular characteristics, have provided quantitative and unbiased identification of distinct neuronal subtypes, when applied to selected datasets. However, better and more robust classification methods are needed for increasingly complex and larger datasets. Here, we explored the use of affinity propagation, a recently developed unsupervised classification algorithm imported from machine learning, which gives a representative example or exemplar for each cluster. As a case study, we applied affinity propagation to a test dataset of 337 interneurons belonging to four subtypes, previously identified based on morphological and physiological characteristics. We found that affinity propagation correctly classified most of the neurons in a blind, non-supervised manner. Affinity propagation outperformed Ward's method, a current standard clustering approach, in classifying the neurons into 4 subtypes. Affinity propagation could therefore be used in future studies to validly classify neurons, as a first step to help reverse engineer neural circuits.

Aptamer-based polyhedral oligomeric silsesquioxane (POSS)-containing hybrid affinity monolith prepared via a "one-pot" process for selective extraction of ochratoxin A.

PubMed

Chen, Yiqiong; Chen, Maolin; Chi, Jinxin; Yu, Xia; Chen, Yongxuan; Lin, Xucong; Xie, Zenghong

2018-08-17

A novel aptamer-based polyhedral oligomeric silsesquioxane (POSS)-containing hybrid affinity monolith has been prepared with a facile "one-pot" process simultaneously via "free radical polymerization" and "thiol-ene" click reaction, and used for on-line selective extraction and practical analysis to trace ochratoxin A (OTA). By using the ternary porogenic mixture composed of water/DMF/PEG, a homogeneous polymerization mixture with POSS chemicals, acrylate-based monomers and aptamer aqueous solution was obtained, and the copolymerization of POSS chemicals, polymer monomers and aptamer aqueous solution was systematically studied. Characterizations such as the morphology, FT-IR and fluorescence spectra, mechanical stability, dynamic binding capacity, cross-reactivity and selectivity of the resultant affinity monolith were also evaluated. Attributed to the porous monolithic structure and aptamer-based affinity interaction, acceptable selective recognition and recovery yields towards trace OTA were obtained. With a 5-fold volume enrichment, the limit of detection (LOD) and limit of quantitation (LOQ) of OTA in fortified beer samples were gained at 0.025 ng/mL (S/N = 3) and 0.045 ng/mL (S/N = 10), respectively. It could be competent for the sensitive measure of actual OTA residues in real beer samples. In comparison with the previously reported strategies containing common "sol-gel" chemistry, the proposed protocol to fabricating aptamer-modified POSS-containing hybrid affinity monolith showed a simpler preparation with acceptable selectivity and higher recovery to trace OTA. Copyright © 2018 Elsevier B.V. All rights reserved.

Boronic acid recognition of non-interacting carbohydrates for biomedical applications: increasing fluorescence signals of minimally interacting aldoses and sucralose†

PubMed Central

Resendez, Angel; Halim, Md Abdul; Singh, Jasmeet; Webb, Dominic-Luc

2017-01-01

To address carbohydrates that are commonly used in biomedical applications with low binding affinities for boronic acid based detection systems, two chemical modification methods were utilized to increase sensitivity. Modified carbohydrates were analyzed using a two component fluorescent probe based on boronic acid-appended viologen–HPTS (4,4′-o-BBV). Carbohydrates normally giving poor signals (fucose, l-rhamnose, xylose) were subjected to sodium borohydride (NaBH4) reduction in ambient conditions for 1 h yielding the corresponding sugar alcohols from fucose, l-rhamnose and xylose in essentially quantitative yields. Compared to original aldoses, apparent binding affinities were increased 4–25-fold. The chlorinated sweetener and colon permeability marker sucralose (Splenda), otherwise undetectable by boronic acids, was dechlorinated to a detectable derivative by reactive oxygen and hydroxide intermediates by the Fenton reaction or by H2O2 and UV light. This method is specific to sucralose as other common sugars, such as sucrose, do not contain any carbon-chlorine bonds. Significant fluorescence response was obtained for chemically modified sucralose with the 4,4′-o-BBV–HPTS probe system. This proof of principle can be applied to biomedical applications, such as gut permeability, malabsorption, etc. PMID:29130464

Boronic acid recognition of non-interacting carbohydrates for biomedical applications: increasing fluorescence signals of minimally interacting aldoses and sucralose.

PubMed

Resendez, Angel; Halim, Md Abdul; Singh, Jasmeet; Webb, Dominic-Luc; Singaram, Bakthan

2017-11-22

To address carbohydrates that are commonly used in biomedical applications with low binding affinities for boronic acid based detection systems, two chemical modification methods were utilized to increase sensitivity. Modified carbohydrates were analyzed using a two component fluorescent probe based on boronic acid-appended viologen-HPTS (4,4'-o-BBV). Carbohydrates normally giving poor signals (fucose, l-rhamnose, xylose) were subjected to sodium borohydride (NaBH 4 ) reduction in ambient conditions for 1 h yielding the corresponding sugar alcohols from fucose, l-rhamnose and xylose in essentially quantitative yields. Compared to original aldoses, apparent binding affinities were increased 4-25-fold. The chlorinated sweetener and colon permeability marker sucralose (Splenda), otherwise undetectable by boronic acids, was dechlorinated to a detectable derivative by reactive oxygen and hydroxide intermediates by the Fenton reaction or by H 2 O 2 and UV light. This method is specific to sucralose as other common sugars, such as sucrose, do not contain any carbon-chlorine bonds. Significant fluorescence response was obtained for chemically modified sucralose with the 4,4'-o-BBV-HPTS probe system. This proof of principle can be applied to biomedical applications, such as gut permeability, malabsorption, etc.

Diverging affinity of tospovirus RNA silencing suppressor proteins, NSs, for various RNA duplex molecules.

PubMed

Schnettler, Esther; Hemmes, Hans; Huismann, Rik; Goldbach, Rob; Prins, Marcel; Kormelink, Richard

2010-11-01

The tospovirus NSs protein was previously shown to suppress the antiviral RNA silencing mechanism in plants. Here the biochemical analysis of NSs proteins from different tospoviruses, using purified NSs or NSs containing cell extracts, is described. The results showed that all tospoviral NSs proteins analyzed exhibited affinity to small double-stranded RNA molecules, i.e., small interfering RNAs (siRNAs) and micro-RNA (miRNA)/miRNA* duplexes. Interestingly, the NSs proteins from tomato spotted wilt virus (TSWV), impatiens necrotic spot virus (INSV), and groundnut ringspot virus (GRSV) also showed affinity to long double-stranded RNA (dsRNA), whereas tomato yellow ring virus (TYRV) NSs did not. The TSWV NSs protein was shown to be capable of inhibiting Dicer-mediated cleavage of long dsRNA in vitro. In addition, it suppressed the accumulation of green fluorescent protein (GFP)-specific siRNAs during coinfiltration with an inverted-repeat-GFP RNA construct in Nicotiana benthamiana. In vivo interference of TSWV NSs in the miRNA pathway was shown by suppression of an enhanced GFP (eGFP) miRNA sensor construct. The ability to stabilize miRNA/miRNA* by different tospovirus NSs proteins in vivo was demonstrated by increased accumulation and detection of both miRNA171c and miRNA171c* in tospovirus-infected N. benthamiana. All together, these data suggest that tospoviruses interfere in the RNA silencing pathway by sequestering siRNA and miRNA/miRNA* molecules before they are uploaded into their respective RNA-induced silencing complexes. The observed affinity to long dsRNA for only a subset of the tospoviruses studied is discussed in light of evolutional divergence and their ancestral relation to the animal-infecting members of the Bunyaviridae.

Investigation on the pH-dependent binding of benzocaine and lysozyme by fluorescence and absorbance.

PubMed

Li, Shihui; Li, Daojin

2011-11-01

The interaction mechanism between benzocaine (BZC) and lysozyme (Lys) has been investigated by fluorescence, synchronous fluorescence, ultraviolet-vis (UV) absorption spectra, and three-dimensional fluorescence (3-D) in various pH medium. The observations of fluorescence spectra were mainly rationalized in terms of a static quenching process at lower concentration of BZC (C(BZC)/C(Lys)<9) and a combined quenching process at higher concentration of BZC (C(BZC)/C(Lys)>9) at pH 7.4 and 8.4. However, the fluorescence quenching was mainly arisen from static quenching by complex formation in all studied drug concentrations at pH 3.5. The structural characteristics of BZC and Lys were probed, and their binding affinities were determined under different pH conditions (pH 3.5, 7.4, and 8.4). The results indicated that the binding abilities of BZC to Lys decreased at the pH below and above the simulative physiological condition (pH 7.4) due to the alterations of the protein secondary and tertiary structures or the structural change of BZC. The effect of BZC on the conformation of Lys was analyzed using UV, synchronous fluorescence and three-dimensional fluorescence under different pH conditions. These results indicate that the binding of BZC to Lys causes apparent change in the secondary and tertiary structures of Lys. The effect of Zn(2+) on the binding constant of BZC with Lys under various pH conditions (pH 3.5, 7.4, and 8.4) was also studied. Copyright © 2011 Elsevier B.V. All rights reserved.

Affinity entrapment of oligosaccharides and glycopeptides using free lectin solution.

PubMed

Yodoshi, Masahiro; Oyama, Takehiro; Masaki, Ken; Kakehi, Kazuaki; Hayakawa, Takao; Suzuki, Shigeo

2011-01-01

Two procedures were proposed for the specific recovery of fluorescent derivatives of glycoprotein-derived oligosaccharides and tryptic glycopeptides using certain plant lectins. The first was based on the salting out of oligosaccharide-lectin conjugates with ammonium sulfate. Oligosaccharides specifically bound to lectins were recovered free from lectins using ethanol precipitation after dissolution in water. This method enabled group separation of 2-aminopyridine-labeled oligosaccharides derived from ovalbumin to galacto-oligosaccharides and agalacto-oligosaccharides by Ricinus communis agglutinin, and to high mannose- and hybrid-type oligosaccharides by wheat-germ agglutinin. Fractional precipitation based on differences in affinity for concanavalin A was accomplished by adding an appropriate concentration of methyl α-mannoside as an inhibitor. In the second method, tryptic digests of glycoproteins were mixed with a lectin solution, and the glycopeptide-lectin conjugates were specifically trapped on a centrifugal ultrafiltration membrane with cut-off of 10 kD. Trapped glycopeptides, as retentates, were passed through membranes by resuspension in diluted acid. This method is particularly useful for the enrichment of glycopeptides in protease digestion mixtures for glycosylation analyses by liquid chromatography-mass spectrometry.

Compound immobilization and drug-affinity chromatography.

PubMed

Rix, Uwe; Gridling, Manuela; Superti-Furga, Giulio

2012-01-01

Bioactive small molecules act through modulating a yet unpredictable number of targets. It is therefore of critical importance to define the cellular target proteins of a compound as an entry point to understanding its mechanism of action. Often, this can be achieved in a direct fashion by chemical proteomics. As with any affinity chromatography, immobilization of the bait to a solid support is one of the earliest and most crucial steps in the process. Interfering with structural features that are important for identification of a target protein will be detrimental to binding affinity. Also, many molecules are sensitive to heat or to certain chemicals, such as acid or base, and might be destroyed during the process of immobilization, which therefore needs to be not only efficient, but also mild. The subsequent affinity chromatography step needs to preserve molecular and conformational integrity of both bait compound and proteins in order to result in the desired specific enrichment while ensuring a high level of compatibility with downstream analysis by mass spectrometry. Thus, the right choice of detergent, buffer, and protease inhibitors is also essential. This chapter describes a widely applicable procedure for the immobilization of small molecule drugs and for drug-affinity chromatography with subsequent protein identification by mass spectrometry.

The effects of pH and temperature on fluorescent calcium indicators as determined with Chelex-100 and EDTA buffer systems.

PubMed

Lattanzio, F A

1990-08-31

A novel method of determining the apparent dissociation constants of fluorescent calcium indicators is described which utilizes Chelex-100 ion exchange resin and 45Ca. The affinity for calcium of indicators fluo-3, fura-2 and indo-1 measured at either 22 degrees or 37 degrees C decreases as pH is decreased from 7.4 to 5.5. These measurements agree with determinations made using EDTA-calcium buffers. The 1:1 calcium:indicator complex is maintained under all conditions. The necessity to correct dissociation constants during intracellular acidification to properly interpret fluorescence measurements is illustrated by indo-1 measurements in the ischemic rat heart.

Synthesis and cell imaging applications of fluorescent mono/di/tri-heterocyclyl-2,6-dicyanoanilines.

PubMed

Pisal, Mahesh M; Annadate, Ritesh A; Athalye, Meghana C; Kumar, Deepak; Chavan, Subhash P; Sarkar, Dhiman; Borate, Hanumant B

2017-02-15

Synthesis of 3,4,5-triheterocyclyl-2,6-dicyanoanilines, starting from heterocyclic aldehydes and 1,2-diheterocycle-substituted ethanones, is described. 2,6-Dicyanoanilines with one or two heterocyclic substituents have also been synthesized. It was found that some of these molecules have selective cell-staining properties useful for cell imaging applications. The compounds 1g, 10f and 11 were found to stain cytoplasm of the cells in contact but not the nucleus while the compound 12 showed affinity to apoptotic cells resulting in blue fluorescence. The cell imaging results with compound 12 were similar to Annexin V-FITC, a known reagent containing recombinant Annexin V conjugated to green-fluorescent FITC dye, used for detection of apoptotic cells. These compounds were found to be non-cytotoxic and have potential application as cell imaging agents. Copyright © 2016 Elsevier Ltd. All rights reserved.

Surfactant-free Colloidal Particles with Specific Binding Affinity

PubMed Central

2017-01-01

Colloidal particles with specific binding affinity are essential for in vivo and in vitro biosensing, targeted drug delivery, and micrometer-scale self-assembly. Key to these techniques are surface functionalizations that provide high affinities to specific target molecules. For stabilization in physiological environments, current particle coating methods rely on adsorbed surfactants. However, spontaneous desorption of these surfactants typically has an undesirable influence on lipid membranes. To address this issue and create particles for targeting molecules in lipid membranes, we present here a surfactant-free coating method that combines high binding affinity with stability at physiological conditions. After activating charge-stabilized polystyrene microparticles with EDC/Sulfo-NHS, we first coat the particles with a specific protein and subsequently covalently attach a dense layer of poly(ethyelene) glycol. This polymer layer provides colloidal stability at physiological conditions as well as antiadhesive properties, while the protein coating provides the specific affinity to the targeted molecule. We show that NeutrAvidin-functionalized particles bind specifically to biotinylated membranes and that Concanavalin A-functionalized particles bind specifically to the glycocortex of Dictyostelium discoideum cells. The affinity of the particles changes with protein density, which can be tuned during the coating procedure. The generic and surfactant-free coating method reported here transfers the high affinity and specificity of a protein onto colloidal polystyrene microparticles. PMID:28847149

Study and selection of in vivo protein interactions by coupling bimolecular fluorescence complementation and flow cytometry.

PubMed

Morell, Montse; Espargaro, Alba; Aviles, Francesc Xavier; Ventura, Salvador

2008-01-01

We present a high-throughput approach to study weak protein-protein interactions by coupling bimolecular fluorescent complementation (BiFC) to flow cytometry (FC). In BiFC, the interaction partners (bait and prey) are fused to two rationally designed fragments of a fluorescent protein, which recovers its function upon the binding of the interacting proteins. For weak protein-protein interactions, the detected fluorescence is proportional to the interaction strength, thereby allowing in vivo discrimination between closely related binders with different affinity for the bait protein. FC provides a method for high-speed multiparametric data acquisition and analysis; the assay is simple, thousands of cells can be analyzed in seconds and, if required, selected using fluorescence-activated cell sorting (FACS). The combination of both methods (BiFC-FC) provides a technically straightforward, fast and highly sensitive method to validate weak protein interactions and to screen and identify optimal ligands in biologically synthesized libraries. Once plasmids encoding the protein fusions have been obtained, the evaluation of a specific interaction, the generation of a library and selection of active partners using BiFC-FC can be accomplished in 5 weeks.

Novel heterocyclic thiosemicarbazones derivatives as colorimetric and "turn on" fluorescent sensors for fluoride anion sensing employing hydrogen bonding.

PubMed

Ashok Kumar, S L; Saravana Kumar, M; Sreeja, P B; Sreekanth, A

2013-09-01

Two novel heterocyclic thiosemicarbazone derivatives have been synthesized, and characterized, by means of spectroscopic and single crystal X-ray diffraction methods. Their chromophoric-fluorogenic response towards anions in competing solvent dimethyl sulfoxide (DMSO) was studied. The receptor shows selective recognition towards fluoride anion. The binding affinity of the receptors with fluoride anion was calculated using UV-visible and fluorescence spectroscopic techniques. Copyright © 2013 Elsevier B.V. All rights reserved.

Fluorescence resonance energy transfer between ZnSe ZnS quantum dots and bovine serum albumin in bioaffinity assays of anticancer drugs

NASA Astrophysics Data System (ADS)

Shu, Chang; Ding, Li; Zhong, Wenying

2014-10-01

In the current work, using ZnSe ZnS quantum dots (QDs) as representative nanoparticles, the affinities of seven anticancer drugs for bovine serum albumin (BSA) were studied using fluorescence resonance energy transfer (FRET). The FRET efficiency of BSA-QD conjugates can reach as high as 24.87% by electrostatic interaction. The higher binding constant (3.63 × 107 L mol-1) and number of binding sites (1.75) between ZnSe ZnS QDs and BSA demonstrated that the QDs could easily associate to plasma proteins and enhance the transport efficacy of drugs. The magnitude of binding constants (103-106 L mol-1), in the presence of QDs, was between drugs-BSA and drugs-QDs in agreement with common affinities of drugs for serum albumins (104-106 L mol-1) in vivo. ZnSe ZnS QDs significantly increased the affinities for BSA of Vorinostat (SAHA), Docetaxel (DOC), Carmustine (BCNU), Doxorubicin (Dox) and 10-Hydroxycamptothecin (HCPT). However, they slightly reduced the affinities of Vincristine (VCR) and Methotrexate (MTX) for BSA. The recent work will not only provide useful information for appropriately understanding the binding affinity and binding mechanism at the molecular level, but also illustrate the ZnSe ZnS QDs are perfect candidates for nanoscal drug delivery system (DDS).

Fluorescence-based strategies to investigate the structure and dynamics of aptamer-ligand complexes

NASA Astrophysics Data System (ADS)

Perez-Gonzalez, Cibran; Lafontaine, Daniel; Penedo, J.

2016-08-01

In addition to the helical nature of double-stranded DNA and RNA, single-stranded oligonucleotides can arrange themselves into tridimensional structures containing loops, bulges, internal hairpins and many other motifs. This ability has been used for more than two decades to generate oligonucleotide sequences, so-called aptamers, that can recognize certain metabolites with high affinity and specificity. More recently, this library of artificially-generated nucleic acid aptamers has been expanded by the discovery that naturally occurring RNA sequences control bacterial gene expression in response to cellular concentration of a given metabolite. The application of fluorescence methods has been pivotal to characterize in detail the structure and dynamics of these aptamer-ligand complexes in solution. This is mostly due to the intrinsic high sensitivity of fluorescence methods and also to significant improvements in solid-phase synthesis, post-synthetic labelling strategies and optical instrumentation that took place during the last decade. In this work, we provide an overview of the most widely employed fluorescence methods to investigate aptamer structure and function by describing the use of aptamers labelled with a single dye in fluorescence quenching and anisotropy assays. The use of 2-aminopurine as a fluorescent analog of adenine to monitor local changes in structure and fluorescence resonance energy transfer (FRET) to follow long-range conformational changes is also covered in detail. The last part of the review is dedicated to the application of fluorescence techniques based on single-molecule microscopy, a technique that has revolutionized our understanding of nucleic acid structure and dynamics. We finally describe the advantages of monitoring ligand-binding and conformational changes, one molecule at a time, to decipher the complexity of regulatory aptamers and summarize the emerging folding and ligand-binding models arising from the application of these

Fluorescence-Based Strategies to Investigate the Structure and Dynamics of Aptamer-Ligand Complexes

PubMed Central

Perez-Gonzalez, Cibran; Lafontaine, Daniel A.; Penedo, J. Carlos

2016-01-01

In addition to the helical nature of double-stranded DNA and RNA, single-stranded oligonucleotides can arrange themselves into tridimensional structures containing loops, bulges, internal hairpins and many other motifs. This ability has been used for more than two decades to generate oligonucleotide sequences, so-called aptamers, that can recognize certain metabolites with high affinity and specificity. More recently, this library of artificially-generated nucleic acid aptamers has been expanded by the discovery that naturally occurring RNA sequences control bacterial gene expression in response to cellular concentration of a given metabolite. The application of fluorescence methods has been pivotal to characterize in detail the structure and dynamics of these aptamer-ligand complexes in solution. This is mostly due to the intrinsic high sensitivity of fluorescence methods and also to significant improvements in solid-phase synthesis, post-synthetic labeling strategies and optical instrumentation that took place during the last decade. In this work, we provide an overview of the most widely employed fluorescence methods to investigate aptamer structure and function by describing the use of aptamers labeled with a single dye in fluorescence quenching and anisotropy assays. The use of 2-aminopurine as a fluorescent analog of adenine to monitor local changes in structure and fluorescence resonance energy transfer (FRET) to follow long-range conformational changes is also covered in detail. The last part of the review is dedicated to the application of fluorescence techniques based on single-molecule microscopy, a technique that has revolutionized our understanding of nucleic acid structure and dynamics. We finally describe the advantages of monitoring ligand-binding and conformational changes, one molecule at a time, to decipher the complexity of regulatory aptamers and summarize the emerging folding and ligand-binding models arising from the application of these

Comparative analysis the binding affinity of mycophenolic sodium and meprednisone with human serum albumin: Insight by NMR relaxation data and docking simulation.

PubMed

Ma, Xiaoli; He, Jiawei; Yan, Jin; Wang, Qing; Li, Hui

2016-03-25

Mycophenolic sodium is an immunosuppressive agent that is always combined administration with corticosteroid in clinical practice. Considering the distribution and side-effect of the drug may change when co-administrated drug exist, this paper comparatively analyzed the binding ability of mycophenolic sodium and meprednisone toward human serum albumin by nuclear magnetic resonance relaxation data and docking simulation. The nuclear magnetic resonance approach was based on the analysis of proton selective and non-selective relaxation rate enhancement of the ligand in the absence and presence of macromolecules. The contribution of the bound ligand fraction to the observed relaxation rate in relation to protein concentration allowed the calculation of the affinity index. This approach allowed the comparison of the binding affinity of mycophenolic sodium and meprednisone. Molecular modeling was operated to simulate the binding model of ligand and albumin through Autodock 4.2.5. Competitive binding of mycophenolic sodium and meprednisone was further conducted through fluorescence spectroscopy. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

5-Arylvinyl-2,2′-bipyridyls: Bright “push–pull” dyes as components in fluorescent indicators for zinc ions

PubMed Central

Zhu, Lei; Younes, Ali H.; Yuan, Zhao; Clark, Ronald J.

2015-01-01

This article reviews the zinc(II)-dependent photophysical properties of arylvinylbipyridines (AVBs), a class of fluoroionophores in which 2,2′-bipyridyl and an aryl moiety are electronically conjugated. Zinc(II) binding of an AVB may lead to an emission bathochromic shift of the fluoroionophore without diminishing its fluorescence quantum yield. This observation can be explained using the excited state model of electron donor–π bridge–electron acceptor “push–pull” fluorophores, in which the bipy moiety acts as an electron acceptor, and zinc(II)-coordination strengthens its electron affinity. The spectral sensitivity of bipy-containing fluoroionophores, such as AVBs, to zinc(II) can be exploited to prepare fluorescent indicators for this ion. In several cases, AVB moieties are incorporated in fluorescent heteroditopic ligands, so that the variation of zinc(II) concentration over a relatively large range can be correlated to fluorescence changes in either intensity or color. AVB fluoroionophores are also used to introduce an intramolecular Förster resonance energy transfer (FRET) strategy for creating zinc(II) indicators with high photostability and a narrow emission band, two desired characteristics of dyes used in fluorescence microscopy. PMID:26190906

A novel fluorescent aptasensor based on gold and silica nanoparticles for the ultrasensitive detection of ochratoxin A

NASA Astrophysics Data System (ADS)

Taghdisi, Seyed Mohammad; Danesh, Noor Mohammad; Beheshti, Hamed Reza; Ramezani, Mohammad; Abnous, Khalil

2016-02-01

Analytical approaches for the detection and quantitation of ochratoxin A (OTA) in blood serum and food products are high in demand. In this study, a fluorescent aptamer-based sensor (aptasensor) is developed for the selective and sensitive detection of OTA, based on a complementary strand of aptamer (CS) and two types of nanoparticles, gold nanoparticles (AuNPs) and silica nanoparticles (SNPs) coated with streptavidin. The fabricated aptasensor inherits the characteristics of SNPs, as enhancers of fluorescence intensity; AuNPs, such as large surface area and unique optical properties; and high affinity of the aptamer toward its target compared to its CS. In the absence of OTA, no FAM and biotin-labeled CS is in the environment of the SNPs coated with streptavidin, which leads to no fluorescence emission. In the presence of the target, an FAM and biotin-labeled CS-SNPs coated with streptavidin conjugate is formed, thus resulting in a very strong fluorescence emission. The designed fluorescent aptasensor exhibits high selectivity toward OTA with a limit of detection (LOD) as low as 0.098 nM. Furthermore, the fabricated aptasensor was successfully applied for the detection of OTA in grape juice and serum with LODs of 0.113 and 0.152 nM, respectively.

Co(II), Ni(II), Cu(II) and Zn(II) complexes of tridentate ONO donor Schiff base ligand: Synthesis, characterization, thermal, non-isothermal kinetics and DFT calculations

NASA Astrophysics Data System (ADS)

Kusmariya, Brajendra S.; Mishra, A. P.

2017-02-01

We report here four mononuclear Co(II), Ni(II), Cu(II) and Zn(II) coordination compounds of general formula [M(L)2] {L = dcp; M = CoII, CuII & ZnII} and [M(L)(H2O)]·H2O {L = dcp; M = NiII} derived from tridentate 2,4-dichloro-6-{[(3-chloro-2-hydroxy-5-nitrophenyl)imino]methyl}phenol (dcp) ligand. These compounds were synthesized and characterized by elemental analysis, FT-IR, uv-vis, 1H NMR, molar conductance, magnetic moment, thermal, PXRD and SEM-EDX. The Powder X-ray Diffraction patterns and SEM analyses showed the crystalline nature of synthesized compounds. The peak broadening was explained in terms of crystallite size and the lattice strain using Scherrer and Williamson-Hall method. Thermogravimetric analysis was performed to determine the thermal stability of synthesized compounds under nitrogen atmosphere up to 820 K at 10 Kmin-1 heating rate. The kinetic and thermodynamic parameters of thermal decomposition were calculated using Coats-Redfern (C-R), Piloyan-Novikova (P-N) and Horowitz-Metzger (H-M) methods assuming first order degradation. The calculated optical band gap values of complexes were found to be in semiconducting range. To support the experimental findings, and derive some fruitful information viz. frequency calculations, HOMO-LUMO, energy gap (ΔE), molecular electrostatic potential (MEP), spin density, absorption spectra etc.; theoretical calculations by means of DFT and TD-DFT at B3LYP level were incorporated.

Complexation equilibria and coordination aspects of Zn(II) complexes contain 2-aminobenzamide and some bioactive amino acid mixed ligands: pH-metric, spectroscopic and thermodynamic studies.

PubMed

Dharmaraja, Jeyaprakash; Subbaraj, Paramasivam; Esakkidurai, Thirugnanasamy; Shobana, Sutha; Raji, Saravanan

2014-01-01

Mixed ligand complexation of 2-aminobenzamide (2AB) as ligand [L] with Zn(II) in the presence of some bio-relevant amino acid constituents like glycine (gly), L-alanine (ala), L-valine (val) and L-phenylalanine (phe) as ligand [B] have been investigated using pH-metric measurements with a combined pH electrode at different temperatures (300, 310, 320 and 330 ± 0.1 K) in 50% (v/v) ethanol-water mixture containing I = 0.15 M NaClO(4) as supporting electrolyte. Computer assisted analysis of the experimental titration data showed the presence of ZnLB and ZnLB2 species as mixed ligand complexes in addition to various binary species. In ZnLB/ZnLB(2) species, both primary and secondary ligands act as bidentate to form a stable six, five membered chelate ring. The calculated stabilization parameter Deltalog K, log X, log X' and % R.S. values clearly show the mixed ligand complexes have higher stabilities than their binary. Thermodynamic parameters DeltaG, DeltaH and DeltaS have been derived from the temperature dependence of the stability constants. The complexation behavior of ZnLB species has been studied by means of electronic spectra. The percentage distribution of various binary and mixed ligand species of each type of the complexes in solution depending on pH and the ratio of Zn(II) to 2-aminobenzamide/amino acid of the systems.

Modern affinity reagents: Recombinant antibodies and aptamers.

PubMed

Groff, Katherine; Brown, Jeffrey; Clippinger, Amy J

2015-12-01

Affinity reagents are essential tools in both basic and applied research; however, there is a growing concern about the reproducibility of animal-derived monoclonal antibodies. The need for higher quality affinity reagents has prompted the development of methods that provide scientific, economic, and time-saving advantages and do not require the use of animals. This review describes two types of affinity reagents, recombinant antibodies and aptamers, which are non-animal technologies that can replace the use of animal-derived monoclonal antibodies. Recombinant antibodies are protein-based reagents, while aptamers are nucleic-acid-based. In light of the scientific advantages of these technologies, this review also discusses ways to gain momentum in the use of modern affinity reagents, including an update to the 1999 National Academy of Sciences monoclonal antibody production report and federal incentives for recombinant antibody and aptamer efforts. In the long-term, these efforts have the potential to improve the overall quality and decrease the cost of scientific research. Copyright © 2015 Elsevier Inc. All rights reserved.

Lanthanide ions induce hydrolysis of hemoglobin-bound 2,3-diphosphoglycerate (2,3-DPG), conformational changes of globin and bidirectional changes of 2,3-DPG-hemoglobin's oxygen affinity.

PubMed

Cheng, Y; Lin, H; Xue, D; Li, R; Wang, K

2001-02-14

The changes in structure and function of 2,3-diphosphoglycerate-hemoglobin (2,3-DPG-Hb) induced by Ln(3+) binding were studied by spectroscopic methods. The binding of lanthanide cations to 2,3-DPG is prior to that to Hb. Ln(3+) binding causes the hydrolysis of either one from the two phosphomonoester bonds in 2,3-DPG non-specifically. The results using the ultrafiltration method indicate that Ln(3+) binding sites for Hb can be classified into three categories: i.e. positive cooperative sites (N(I)), non-cooperative strong sites (N(S)) and non-cooperative weak sites (N(W)) with binding constants in decreasing order: K(I)>K(S)>K(W). The total number of binding sites amounts to about 65 per Hb tetramer. Information on reaction kinetics was obtained from the change of intrinsic fluorescence in Hb monitored by stopped-flow fluorometry. Fluctuation of fluorescence dependent on Ln(3+) concentration and temperature was observed and can be attributed to the successive conformational changes induced by Ln(3+) binding. The results also reveal the bidirectional changes of the oxygen affinity of Hb in the dependence on Ln(3+) concentration. At the range of [Ln(3+)]/[Hb]<2, the marked increase of oxygen affinity (P(50) decrease) with the Ln(3+) concentration can be attributed to the hydrolysis of 2,3-DPG, while the slight rebound of oxygen affinity in higher Ln(3+) concentration can be interpreted by the transition to the T-state of the Hb tetramer induced by Ln(3+) binding. This was indicated by the changes in secondary structure characterized by the decrease of alpha-helix content.

Synthesis and preliminary biological evaluations of fluorescent or 149Promethium labeled Trastuzumab-polyethylenimine

DOE PAGES

Fitzsimmons, Jonathan; Nayak, Tapan; Cutler, Cathy; ...

2015-12-30

Radioimmunotherapy utilize a targeting antibody coupled to a therapeutic isotope to target and treat a tumor or disease. In this study we examine the synthesis and cell binding of a polymer scaffold containing a radiotherapeutic isotope and a targeting antibody. Methods: The multistep synthesis of a fluorescent or 149Promethium-labeled Trastuzumab-polyethyleneimine (PEI), Trastuzumab, or PEI is described. In vitro uptake, internalization and/or the binding affinity to the Her2/neu expressing human breast adenocarcinoma SKBr3 cells was investigated with the labeled compounds. Fluorescent-labeled Trastuzumab-PEI was internalized more into cells at 2 and 18 h than fluorescent-labeled Trastuzumab or PEI. The fluorescent-labeled Trastuzumab wasmore » concentrated on the cell surface at 2 and 18 h and the labeled PEI had minimal uptake. DOTA-PEI was prepared and contained an average of 16 chelates per PEI; the compound was radio-labeled with 149Promethium and conjugated to Trastuzumab. The purified 149Pm-DOTA-PEI-Trastuzumab had a radiochemical purity of 96.7% and a specific activity of 0.118 TBq/g. The compound demonstrated a dissociation constant for the Her2/neu receptor of 20.30 ± 6.91 nM. In conclusion, the results indicate the DOTA-PEI-Trastuzumab compound has potential as a targeted therapeutic carrier, and future in vivo studies should be performed.« less

Affinity Proteomics in the mountains: Alpbach 2015.

PubMed

Taussig, Michael J

2016-09-25

The 2015 Alpbach Workshop on Affinity Proteomics, organised by the EU AFFINOMICS consortium, was the 7th workshop in this series. As in previous years, the focus of the event was the current state of affinity methods for proteome analysis, including complementarity with mass spectrometry, progress in recombinant binder production methods, alternatives to classical antibodies as affinity reagents, analysis of proteome targets, industry focus on biomarkers, and diagnostic and clinical applications. The combination of excellent science with Austrian mountain scenery and winter sports engender an atmosphere that makes this series of workshops exceptional. The articles in this Special Issue represent a cross-section of the presentations at the 2015 meeting. Copyright © 2016 Elsevier B.V. All rights reserved.

EGFR-directed Affibody for fluorescence-guided glioma surgery: time-dose analysis (Conference Presentation)

NASA Astrophysics Data System (ADS)

Ribeiro de Souza, Ana Luiza; Marra, Kayla; Gunn, Jason R.; Elliott, Jonathan T.; Samkoe, Kimberley S.; Paulsen, Keith D.; Draney, Daniel R.; Feldwisch, Joachim

2016-03-01

The key to fluorescence guided surgical oncology is the ability to create specific contrast between normal and glioma tissue. The blood brain barrier that limits the delivery of substances to the normal brain is broken in tumors, allowing accumulation of agents in the tumor interior. However, for a clinical success, imaging agents should be in the infiltrative edges to minimize the resection of normal brain while enable the removal of tumor. The aberrant overexpression and/or activation of EGFR is associated with many types of cancers, including glioblastoma and the injection of a fluorescent molecule targeted to these receptors would improve tumor contrast during fluorescence guided surgery. Affibody molecules have intentional medium affinity and high potential specificity, which are the desirable features of a good surgical imaging agent. The aim of this study was evaluate the brain/glioma uptake of ABY029 labeled with near-infrared dye IRDye800CW after intravenous injection. Rats were either inoculated with orthotopic implantations of U251 human glioma cell line or PBS (shams control) in the brain. The tumors were allowed to grow for 2-3 weeks before carrying out fluorescent tracer experiments. Fluorescent imaging of ex vivo brain slices from rats was acquired at different time points after infection of fluorescently labeled EGFR-specific affibody to verify which time provided maximal contrast tumor to normal brain. Although the tumor was most clearly visualized after 1h of IRDye800CW-labeled ABY029 injection, the tumor location could be identified from the background after 48h. These results suggest that the NIR-labeled affibody examined shows excellent potential to increase surgical visualization for confirmed EGFR positive tumors.

Direct Measurement of Equilibrium Constants for High-Affinity Hemoglobins

PubMed Central

Kundu, Suman; Premer, Scott A.; Hoy, Julie A.; Trent, James T.; Hargrove, Mark S.

2003-01-01

The biological functions of heme proteins are linked to their rate and affinity constants for ligand binding. Kinetic experiments are commonly used to measure equilibrium constants for traditional hemoglobins comprised of pentacoordinate ligand binding sites and simple bimolecular reaction schemes. However, kinetic methods do not always yield reliable equilibrium constants with more complex hemoglobins for which reaction mechanisms are not clearly understood. Furthermore, even where reaction mechanisms are clearly understood, it is very difficult to directly measure equilibrium constants for oxygen and carbon monoxide binding to high-affinity (KD ≪ 1 μM) hemoglobins. This work presents a method for direct measurement of equilibrium constants for high-affinity hemoglobins that utilizes a competition for ligands between the "target" protein and an array of "scavenger" hemoglobins with known affinities. This method is described for oxygen and carbon monoxide binding to two hexacoordinate hemoglobins: rice nonsymbiotic hemoglobin and Synechocystis hemoglobin. Our results demonstrate that although these proteins have different mechanisms for ligand binding, their affinities for oxygen and carbon monoxide are similar. Their large affinity constants for oxygen, 285 and ∼100 μM−1 respectively, indicate that they are not capable of facilitating oxygen transport. PMID:12770899

Affine.m—Mathematica package for computations in representation theory of finite-dimensional and affine Lie algebras

NASA Astrophysics Data System (ADS)

Nazarov, Anton

2012-11-01

In this paper we present Affine.m-a program for computations in representation theory of finite-dimensional and affine Lie algebras and describe implemented algorithms. The algorithms are based on the properties of weights and Weyl symmetry. Computation of weight multiplicities in irreducible and Verma modules, branching of representations and tensor product decomposition are the most important problems for us. These problems have numerous applications in physics and we provide some examples of these applications. The program is implemented in the popular computer algebra system Mathematica and works with finite-dimensional and affine Lie algebras. Catalogue identifier: AENA_v1_0 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AENB_v1_0.html Program obtainable from: CPC Program Library, Queen’s University, Belfast, UK Licensing provisions: Standard CPC licence, http://cpc.cs.qub.ac.uk/licence/licence.html No. of lines in distributed program, including test data, etc.: 24 844 No. of bytes in distributed program, including test data, etc.: 1 045 908 Distribution format: tar.gz Programming language: Mathematica. Computer: i386-i686, x86_64. Operating system: Linux, Windows, Mac OS, Solaris. RAM: 5-500 Mb Classification: 4.2, 5. Nature of problem: Representation theory of finite-dimensional Lie algebras has many applications in different branches of physics, including elementary particle physics, molecular physics, nuclear physics. Representations of affine Lie algebras appear in string theories and two-dimensional conformal field theory used for the description of critical phenomena in two-dimensional systems. Also Lie symmetries play a major role in a study of quantum integrable systems. Solution method: We work with weights and roots of finite-dimensional and affine Lie algebras and use Weyl symmetry extensively. Central problems which are the computations of weight multiplicities, branching and fusion coefficients are solved using one general recurrent

Fluorescence Imaging Study of Extracellular Zinc at the Hippocampal Mossy Fiber Synapse

PubMed Central

Bastian, Chinthasagar; Li, Yang V

2010-01-01

Although synaptically-released, vesicular Zn2+ has been proposed to play a neuromodulatory or neuronal signaling role at the mossy fiber-CA3 synapse, Zn2+ release remains controversial, especially when detected using fluorescent imaging. In the present study, we investigated synaptically released Zn2+ at the mossy fiber synapse in rat hippocampal slices using three chemically distinct, fluorescent Zn2+ indicators. The indicators employed for this study were cell membrane impermeable (or extracellular) Newport Green (KD Zn2+ ~ 1 μM), Zinpyr-4 (KD Zn2+ ~ 1 nM) and FluoZin-3 (KD Zn2+ ~ 15 nM), chosen, in part, for their distinct dissociation constants. Among the three indicators, FluoZin-3 was also sensitive to Ca2+ (KD Ca2+ ~ 100 μM) which was present in the extracellular medium ([Ca2+]o > 2mM). Hippocampal slices loaded with either Newport Green or FluoZin-3 showed increases in fluorescence after electrical stimulation of the mossy fiber pathway. These results are consistent with previous studies suggesting the presence of synaptically-released Zn2+ in the extracellular space during neuronal activities; however, the rise in FluoZin-3 fluorescence observed was complicated by the data that the addition of exogenous Zn2+ onto FluoZin-3 loaded slices gave little change in fluorescence. In the slices loaded with the high-affinity indicator Zinpyr-4, there was little change in fluorescence after mossy fiber activation by electrical stimulation. Further study revealed that the sensitivity of Zinpyr-4 was mitigated by saturation with Zn2+ contamination from the slice. These data suggest that the sensitivity and selectivity of a probe may affect individual outcomes in a given experimental system. PMID:17485170

Rapid and sensitive detection of ketamine in blood using novel fluorescence genosensor.

PubMed

Ding, Yanjun; Li, Xingmei; Guo, Yadong; Yan, Jie; Ling, Jiang; Li, Weichen; Lan, Lingmei; Chang, Yunfeng; Cai, Jifeng; Zha, Lagabaiyla

2017-12-01

In recent years, drug abuse has been considered as a most challenging social problem that aroused public attention. Ketamine has increased in unregulated use as a 'recreational drug' in teenagers. However, there is no suitable and maneuverable detection method for ketamine in situ at the moment. Fluorescence sensor technique, with predominant recognition and simple operation, is a good potential application in drug detection. Here, we first reported a highly sensitive and selective fluorescence genosensor for rapid detection of ketamine based on DNA-templated silver nanoclusters (DNA-AgNCs) probes, in which the DNA sequence could specially recognize ketamine with high affinity. Parameters affecting detection efficiency were investigated and optimized. Under optimum conditions, the as-prepared genosensor can allow for the determination of ketamine in the concentration range of 0.0001-20 μg/mL with two linear equations: one is y = 2.84x-7.139 (R 2 = 0.987) for 0.0001-0.1 μg/mL, and the other is y = 1.87x-0.091 (R 2 = 0.962) for 0.1-20 μg/mL, and the estimated detection limit of ketamine is 0.06 ng/mL. Moreover, the feasibility of this proposed method was also demonstrated by analyzing forensic blood samples. Compared with official gas chromatography/mass spectrometry (GC/MS), this fluorescence genosensor is simple, rapid, and accurate for quantitative determination of ketamine in blood for pharmaceutical and forensic analysis. Overall, it is the first report on a fluorescence genosensor for detecting ketamine directly in blood. This research may provide a new insight for the analyst to band fluorescence genosensor technology together with drug monitoring in the battle against drug abuse and forensic examination. Graphical abstract High selectively detection of ketamine using a novel fluorescence genosensor based on DNA-AgNCs probe.

Hydride affinity scale of various substituted arylcarbeniums in acetonitrile.

PubMed

Zhu, Xiao-Qing; Wang, Chun-Hua

2010-12-23

Combined with the integral equation formalism polarized continuum model (IEFPCM), the hydride affinities of 96 various acylcarbenium ions in the gas phase and CH(3)CN were estimated by using the B3LYP/6-31+G(d)//B3LYP/6-31+G(d), B3LYP/6-311++G(2df,2p)//B3LYP/6-31+G(d), and BLYP/6-311++G(2df,2p)//B3LYP/6-31+G(d) methods for the first time. The results show that the combination of the BLYP/6-311++G(2df,2p)//B3LYP/6-31+G(d) method and IEFPCM could successfully predict the hydride affinities of arylcarbeniums in MeCN with a precision of about 3 kcal/mol. On the basis of the calculated results from the BLYP method, it can be found that the hydride affinity scale of the 96 arylcarbeniums in MeCN ranges from -130.76 kcal/mol for NO(2)-PhCH(+)-CN to -63.02 kcal/mol for p-(Me)(2)N-PhCH(+)-N(Me)(2), suggesting most of the arylcarbeniums are good hydride acceptors. Examination of the effect of the number of phenyl rings attached to the carbeniums on the hydride affinities shows that the increase of the hydride affinities takes place linearly with increasing number of benzene rings in the arylcarbeniums. Analyzing the effect of the substituents on the hydride affinities of arylcarbeniums indicates that electron-donating groups decrease the hydride affinities and electron-withdrawing groups show the opposite effect. The hydride affinities of arylcarbeniums are linearly dependent on the sum of the Hammett substituent parameters σ(p)(+). Inspection of the correlation of the solution-phase hydride affinities with gas-phase hydride affinities and aqueous-phase pK(R)(+) values reveals a remarkably good correspondence of ΔG(H(-)A)(R(+)) with both the gas-phase relative hydride affinities only if the α substituents X have no large electron-donating or -withdrawing properties and the pK(R)(+) values even though the media are dramatically different. The solution-phase hydride affinities also have a linear relationship with the electrophilicity parameter E, and this dependence can

Fluorescent rhodanine-3-acetic acids visualize neurofibrillary tangles in Alzheimer's disease brains.

PubMed

Anumala, Upendra Rao; Gu, Jiamin; Lo Monte, Fabio; Kramer, Thomas; Heyny-von Haußen, Roland; Hölzer, Jana; Goetschy-Meyer, Valerie; Schön, Christian; Mall, Gerhard; Hilger, Ingrid; Czech, Christian; Herms, Jochen; Schmidt, Boris

2013-09-01

There is a high demand for the development of an imaging agent for neurofibrillary tangles (NFTs) detection in Alzheimer's diagnosis. In the present study, a series of rhodanine-3-acetic acids was synthesized and evaluated for fluorescence imaging of NFTs in brain tissues of AD patients. Five out of seven probes have shown excellent binding affinity to NFTs over amyloid plaques in the Thiazine red R displacement assay. However, the selectivity in this in vitro assay is not confirmed by the histopathological evaluation, which indicates significant differences in the binding sites in the assays. Probe 6 showed binding affinity (IC50=19nM) to tau aggregates which is the highest among this series. Probes 2, 3, 4 and 5 display IC50 values of lower than 100nM to tau aggregates to displace Thiazine red R. Evaluation of the cytotoxicity of these five probes with human liver carcinoma cells revealed that these compounds excert negligible cytotoxicity. The in vivo studies with zebrafish embryos confirmed negligible cytotoxicity at 24 and 72h post fertilization. Copyright © 2013 Elsevier Ltd. All rights reserved.

Affine group formulation of the Standard Model coupled to gravity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chou, Ching-Yi, E-mail: l2897107@mail.ncku.edu.tw; Ita, Eyo, E-mail: ita@usna.edu; Soo, Chopin, E-mail: cpsoo@mail.ncku.edu.tw

In this work we apply the affine group formalism for four dimensional gravity of Lorentzian signature, which is based on Klauder’s affine algebraic program, to the formulation of the Hamiltonian constraint of the interaction of matter and all forces, including gravity with non-vanishing cosmological constant Λ, as an affine Lie algebra. We use the hermitian action of fermions coupled to gravitation and Yang–Mills theory to find the density weight one fermionic super-Hamiltonian constraint. This term, combined with the Yang–Mills and Higgs energy densities, are composed with York’s integrated time functional. The result, when combined with the imaginary part of themore » Chern–Simons functional Q, forms the affine commutation relation with the volume element V(x). Affine algebraic quantization of gravitation and matter on equal footing implies a fundamental uncertainty relation which is predicated upon a non-vanishing cosmological constant. -- Highlights: •Wheeler–DeWitt equation (WDW) quantized as affine algebra, realizing Klauder’s program. •WDW formulated for interaction of matter and all forces, including gravity, as affine algebra. •WDW features Hermitian generators in spite of fermionic content: Standard Model addressed. •Constructed a family of physical states for the full, coupled theory via affine coherent states. •Fundamental uncertainty relation, predicated on non-vanishing cosmological constant.« less

A cooperative-binding split aptamer assay for rapid, specific and ultra-sensitive fluorescence detection of cocaine in saliva† †Electronic supplementary information (ESI) available: Optimization of Mg2+ and ATMND concentrations for our CBSA-based ATMND-binding assay; ATMND-reported calibration curve for CBSA-5325 at various cocaine concentrations; ATMND binding affinity for the cocaine-assembled CBSA-5325; K D of 38-GC and different 38-GC mutants for cocaine as characterized by ITC; stem length effects on cocaine-induced CBSA assembly; spectra of CBSA-5335-based fluorescence detection of cocaine in 1× binding buffer; characterization of cocaine binding affinity of CBSA-5335 and PSA using ITC; fluorescence detection of cocaine in saliva with our fluorophore/quencher modified CBSA-5335; calibration curve of our CBSA-5335-based fluorophore/quencher assay in 1× binding buffer and 10% saliva at cocaine concentrations ranging from 0 to 10 μM; bias and precision of the CBSA-5335-based fluorophore/quencher assay; comparison of amplification-free split-aptamer assays for cocaine detection; sequence ID and DNA sequences used in this work. See DOI: 10.1039/c6sc01833e Click here for additional data file.

PubMed Central

Yu, Haixiang; Canoura, Juan; Guntupalli, Bhargav; Lou, Xinhui

2017-01-01

Sensors employing split aptamers that reassemble in the presence of a target can achieve excellent specificity, but the accompanying reduction of target affinity mitigates any overall gains in sensitivity. We for the first time have developed a split aptamer that achieves enhanced target-binding affinity through cooperative binding. We have generated a split cocaine-binding aptamer that incorporates two binding domains, such that target binding at one domain greatly increases the affinity of the second domain. We experimentally demonstrate that the resulting cooperative-binding split aptamer (CBSA) exhibits higher target binding affinity and is far more responsive in terms of target-induced aptamer assembly compared to the single-domain parent split aptamer (PSA) from which it was derived. We further confirm that the target-binding affinity of our CBSA can be affected by the cooperativity of its binding domains and the intrinsic affinity of its PSA. To the best of our knowledge, CBSA-5335 has the highest cocaine affinity of any split aptamer described to date. The CBSA-based assay also demonstrates excellent performance in target detection in complex samples. Using this CBSA, we achieved specific, ultra-sensitive, one-step fluorescence detection of cocaine within fifteen minutes at concentrations as low as 50 nM in 10% saliva without signal amplification. This limit of detection meets the standards recommended by the European Union's Driving under the Influence of Drugs, Alcohol and Medicines program. Our assay also demonstrates excellent reproducibility of results, confirming that this CBSA-platform represents a robust and sensitive means for cocaine detection in actual clinical samples. PMID:28451157

A novel near-infrared fluorescent probe for sensitive detection of β-galactosidase in living cells.

PubMed

Zhang, Jingtuo; Li, Cong; Dutta, Colina; Fang, Mingxi; Zhang, Shuwei; Tiwari, Ashutosh; Werner, Thomas; Luo, Fen-Tair; Liu, Haiying

2017-05-22

A novel near-infrared fluorescent probe for β-galactosidase has been developed based on a hemicyanine skeleton, which is conjugated with a d-galactose residue via a glycosidic bond. The probe serves as a substrate of β-galactosidase and displays rapid and sensitive turn-on fluorescent responses to β-galactosidase in aqueous solution. A 12.8-fold enhancement of fluorescence intensity at 703 nm was observed after incubation of 10 nM of β-galactosidase with 5 μM probe for 10 min. The probe can sensitively detect as little as 0.1 nM of β-galactosidase and shows linear responses to the enzyme concentration below 1.4 nM. The kinetic study showed that the probe has high binding affinity to β-galactosidase with K m  = 3.6 μM. The probe was used to detect β-galactosidase in living cells by employing the premature cell senescence model. The probe exhibited strong fluorescent signals in senescent cells but not in normal cells, which demonstrates that the probe is able to detect the endogenous senescence-associated β-galactosidase in living cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Smart near-infrared fluorescence probes with donor-acceptor structure for in vivo detection of β-amyloid deposits.

PubMed

Cui, Mengchao; Ono, Masahiro; Watanabe, Hiroyuki; Kimura, Hiroyuki; Liu, Boli; Saji, Hideo

2014-03-05

The deposition of β-amyloid (Aβ) plaques in the parenchymal and cortical brain is accepted as the main pathological hallmark of Alzheimer's disease (AD); however, early detection of AD still presents a challenge. With the assistance of molecular imaging techniques, imaging agents specifically targeting Aβ plaques in the brain may lead to the early diagnosis of AD. Herein, we report the design, synthesis, and evaluation of a series of smart near-infrared fluorescence (NIRF) imaging probes with donor-acceptor architecture bridged by a conjugated π-electron chain for Aβ plaques. The chemical structure of these NIRF probes is completely different from Congo Red and Thioflavin-T. Probes with a longer conjugated π system (carbon-carbon double bond) displayed maximum emission in PBS (>650 nm), which falls in the best range for NIRF probes. These probes were proved to have affinity to Aβ plaques in fluorescent staining of brain sections from an AD patient and double transgenic mice, as well as in an in vitro binding assay using Aβ(1-42) aggregates. One probe with high affinity (K(i) = 37 nM, K(d) = 27 nM) was selected for in vivo imaging. It can penetrate the blood-brain barrier of nude mice efficiently and is quickly washed out of the normal brain. Moreover, after intravenous injection of this probe, 22-month-old APPswe/PSEN1 mice exhibited a higher relative signal than control mice over the same period of time, and ex vivo fluorescent observations confirmed the existence of Aβ plaques. In summary, this probe meets most of the requirements for a NIRF contrast agent for the detection of Aβ plaques both in vitro and in vivo.

Cytidine-stabilized gold nanocluster as a fluorescence turn-on and turn-off probe for dual functional detection of Ag(+) and Hg(2+).

PubMed

Zhang, Yuanyuan; Jiang, Hui; Wang, Xuemei

2015-04-22

In this study, we have developed a label-free, dual functional detection strategy for highly selective and sensitive determination of aqueous Ag(+) and Hg(2+) by using cytidine stabilized Au NCs and AuAg NCs as fluorescent turn-on and turn off probes, respectively. The Au NCs and AuAg NCs showed a remarkably rapid response and high selectivity for Ag(+) and Hg(2+) over other metal ions, and relevant detection limit of Ag(+) and Hg(2+) is ca. 10 nM and 30 nM, respectively. Importantly, the fluorescence enhanced Au NCs by doping Ag(+) can be conveniently reusable for the detection of Hg(2+) based on the corresponding fluorescence quenching. The sensing mechanism was based on the high-affinity metallophilic Hg(2+)-Ag(+) interaction, which effectively quenched the fluorescence of AuAg NCs. Furthermore, these fluorescent nanoprobes could be readily applied to Ag(+) and Hg(2+) detection in environmental water samples, indicating their possibility to be utilized as a convenient, dual functional, rapid response, and label-free fluorescence sensor for related environmental and health monitoring. Copyright © 2015 Elsevier B.V. All rights reserved.

Characterization of 5-HT₁A receptors and their complexes with G-proteins in budded baculovirus particles using fluorescence anisotropy of Bodipy-FL-NAN-190.

PubMed

Tõntson, Lauri; Kopanchuk, Sergei; Rinken, Ago

2014-02-01

Bodipy-FL-NAN-190 was found to be well suited for characterization of ligand binding to 5-HT1A receptors expressed in budded baculovirus particles, as binding is accompanied by large increases in fluorescence intensity and anisotropy. This ligand appears to bind rapidly (t1/2,ass<1 min), reversibly (t1/2,diss∼6 min) and has high affinity (Kd=0.30 ± 0.13 nM). This fluorescence anisotropy assay based on Bodipy-FL-NAN-190 binding to baculovirus particles was also a suitable assay system for the pharmacological characterization of non-labelled serotonergic ligands, as well as being sensitive to the presence of G-proteins and guanine nucleotides. Coexpression of αi subunits of human G-proteins in baculovirus particles resulted in the appearance of significantly greater proportion of nucleotide sensitive high affinity agonist binding sites. There were no significant differences between αi1 and αi3 subtypes, while ligand binding in the presence of αi2 had higher sensitivity to GDP and Mn(2+). Copyright © 2014 Elsevier Ltd. All rights reserved.

N-Glycosylation of Asparagine 130 in the Extracellular Domain of the Human Calcitonin Receptor Significantly Increases Peptide Hormone Affinity.

PubMed

Lee, Sang-Min; Booe, Jason M; Gingell, Joseph J; Sjoelund, Virginie; Hay, Debbie L; Pioszak, Augen A

2017-07-05

The calcitonin receptor (CTR) is a class B G protein-coupled receptor that is activated by the peptide hormones calcitonin and amylin. Calcitonin regulates bone remodeling through CTR, whereas amylin regulates blood glucose and food intake by activating CTR in complex with receptor activity-modifying proteins (RAMPs). These receptors are targeted clinically for the treatment of osteoporosis and diabetes. Here, we define the role of CTR N-glycosylation in hormone binding using purified calcitonin and amylin receptor extracellular domain (ECD) glycoforms and fluorescence polarization/anisotropy and isothermal titration calorimetry peptide-binding assays. N-Glycan-free CTR ECD produced in Escherichia coli exhibited ∼10-fold lower peptide affinity than CTR ECD produced in HEK293T cells, which yield complex N-glycans, or in HEK293S GnTI - cells, which yield core N-glycans (Man 5 GlcNAc 2 ). PNGase F-catalyzed removal of N-glycans at N73, N125, and N130 in the CTR ECD decreased peptide affinity ∼10-fold, whereas Endo H-catalyzed trimming of the N-glycans to single GlcNAc residues had no effect on peptide binding. Similar results were observed for an amylin receptor RAMP2-CTR ECD complex. Characterization of peptide-binding affinities of purified N → Q CTR ECD glycan site mutants combined with PNGase F and Endo H treatment strategies and mass spectrometry to define the glycan species indicated that a single GlcNAc residue at CTR N130 was responsible for the peptide affinity enhancement. Molecular modeling suggested that this GlcNAc functions through an allosteric mechanism rather than by directly contacting the peptide. These results reveal an important role for N-linked glycosylation in the peptide hormone binding of a clinically relevant class B GPCR.

Calculation of protein-ligand binding affinities.

PubMed

Gilson, Michael K; Zhou, Huan-Xiang

2007-01-01

Accurate methods of computing the affinity of a small molecule with a protein are needed to speed the discovery of new medications and biological probes. This paper reviews physics-based models of binding, beginning with a summary of the changes in potential energy, solvation energy, and configurational entropy that influence affinity, and a theoretical overview to frame the discussion of specific computational approaches. Important advances are reported in modeling protein-ligand energetics, such as the incorporation of electronic polarization and the use of quantum mechanical methods. Recent calculations suggest that changes in configurational entropy strongly oppose binding and must be included if accurate affinities are to be obtained. The linear interaction energy (LIE) and molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) methods are analyzed, as are free energy pathway methods, which show promise and may be ready for more extensive testing. Ultimately, major improvements in modeling accuracy will likely require advances on multiple fronts, as well as continued validation against experiment.

In Vivo Fluorescence Lifetime Imaging Monitors Binding of Specific Probes to Cancer Biomarkers

PubMed Central

Ardeshirpour, Yasaman; Chernomordik, Victor; Zielinski, Rafal; Capala, Jacek; Griffiths, Gary; Vasalatiy, Olga; Smirnov, Aleksandr V.; Knutson, Jay R.; Lyakhov, Ilya; Achilefu, Samuel; Gandjbakhche, Amir; Hassan, Moinuddin

2012-01-01

One of the most important factors in choosing a treatment strategy for cancer is characterization of biomarkers in cancer cells. Particularly, recent advances in Monoclonal Antibodies (MAB) as primary-specific drugs targeting tumor receptors show that their efficacy depends strongly on characterization of tumor biomarkers. Assessment of their status in individual patients would facilitate selection of an optimal treatment strategy, and the continuous monitoring of those biomarkers and their binding process to the therapy would provide a means for early evaluation of the efficacy of therapeutic intervention. In this study we have demonstrated for the first time in live animals that the fluorescence lifetime can be used to detect the binding of targeted optical probes to the extracellular receptors on tumor cells in vivo. The rationale was that fluorescence lifetime of a specific probe is sensitive to local environment and/or affinity to other molecules. We attached Near-InfraRed (NIR) fluorescent probes to Human Epidermal Growth Factor 2 (HER2/neu)-specific Affibody molecules and used our time-resolved optical system to compare the fluorescence lifetime of the optical probes that were bound and unbound to tumor cells in live mice. Our results show that the fluorescence lifetime changes in our model system delineate HER2 receptor bound from the unbound probe in vivo. Thus, this method is useful as a specific marker of the receptor binding process, which can open a new paradigm in the “image and treat” concept, especially for early evaluation of the efficacy of the therapy. PMID:22384092

GFP tagging of sieve element occlusion (SEO) proteins results in green fluorescent forisomes.

PubMed

Pélissier, Hélène C; Peters, Winfried S; Collier, Ray; van Bel, Aart J E; Knoblauch, Michael

2008-11-01

Forisomes are Ca(2+)-driven, ATP-independent contractile protein bodies that reversibly occlude sieve elements in faboid legumes. They apparently consist of at least three proteins; potential candidates have been described previously as 'FOR' proteins. We isolated three genes from Medicago truncatula that correspond to the putative forisome proteins and expressed their green fluorescent protein (GFP) fusion products in Vicia faba and Glycine max using the composite plant methodology. In both species, expression of any of the constructs resulted in homogenously fluorescent forisomes that formed sieve tube plugs upon stimulation; no GFP fluorescence occurred elsewhere. Isolated fluorescent forisomes reacted to Ca(2+) and chelators by contraction and expansion, respectively, and did not lose fluorescence in the process. Wild-type forisomes showed no affinity for free GFP in vitro. The three proteins shared numerous conserved motifs between themselves and with hypothetical proteins derived from the genomes of M. truncatula, Vitis vinifera and Arabidopsis thaliana. However, they showed neither significant similarities to proteins of known function nor canonical metal-binding motifs. We conclude that 'FOR'-like proteins are components of forisomes that are encoded by a well-defined gene family with relatives in taxa that lack forisomes. Since the mnemonic FOR is already registered and in use for unrelated genes, we suggest the acronym SEO (sieve element occlusion) for this family. The absence of binding sites for divalent cations suggests that the Ca(2+) binding responsible for forisome contraction is achieved either by as yet unidentified additional proteins, or by SEO proteins through a novel, uncharacterized mechanism.

GFP Tagging of Sieve Element Occlusion (SEO) Proteins Results in Green Fluorescent Forisomes

PubMed Central

Pélissier, Hélène C.; Peters, Winfried S.; Collier, Ray; van Bel, Aart J. E.; Knoblauch, Michael

2008-01-01

Forisomes are Ca2+-driven, ATP-independent contractile protein bodies that reversibly occlude sieve elements in faboid legumes. They apparently consist of at least three proteins; potential candidates have been described previously as ‘FOR’ proteins. We isolated three genes from Medicago truncatula that correspond to the putative forisome proteins and expressed their green fluorescent protein (GFP) fusion products in Vicia faba and Glycine max using the composite plant methodology. In both species, expression of any of the constructs resulted in homogenously fluorescent forisomes that formed sieve tube plugs upon stimulation; no GFP fluorescence occurred elsewhere. Isolated fluorescent forisomes reacted to Ca2+ and chelators by contraction and expansion, respectively, and did not lose fluorescence in the process. Wild-type forisomes showed no affinity for free GFP in vitro. The three proteins shared numerous conserved motifs between themselves and with hypothetical proteins derived from the genomes of M. truncatula, Vitis vinifera and Arabidopsis thaliana. However, they showed neither significant similarities to proteins of known function nor canonical metal-binding motifs. We conclude that ‘FOR’-like proteins are components of forisomes that are encoded by a well-defined gene family with relatives in taxa that lack forisomes. Since the mnemonic FOR is already registered and in use for unrelated genes, we suggest the acronym SEO (sieve element occlusion) for this family. The absence of binding sites for divalent cations suggests that the Ca2+ binding responsible for forisome contraction is achieved either by as yet unidentified additional proteins, or by SEO proteins through a novel, uncharacterized mechanism. PMID:18784195

Near infrared fluorescent trypsin stabilized gold nanoclusters as surface plasmon enhanced energy transfer biosensor and in vivo cancer imaging bioprobe.

PubMed

Liu, Jing-Min; Chen, Jia-Tong; Yan, Xiu-Ping

2013-03-19

The simplicity of the green-synthesized routine and the availability of surface modification of diverse bioactive molecules make noble metal nanostructures highly suitable as multifunctional biomaterials for biological and biomedical application. Here, we report the preparation of trypsin stabilized gold nanoclusters (try-AuNCs) with near-infrared fluorescence for biosensing heparin based on surface plasmon enhanced energy transfer (SPEET) and folic acid (FA) modified try-AuNCs for in vivo cancer bioimaging. The SPEET/try-AuNCs fluorescence biosensor was designed via heparin mediated energy transfer between try-AuNCs and cysteamine modified gold nanoparticles (cyst-AuNPs). The developed SPEET/try-AuNCs fluorescence biosensor allowed sensitive and selective detection of heparin with a linear range of 0.1-4.0 μg mL(-1) and a detection limit (3s) of 0.05 μg mL(-1). The relative standard deviation for eleven replicate detections of 2.5 μg mL(-1) heparin was 1.1%, and the recoveries of the spiked heparin in human serum samples ranged from 97% to 100%. In addition, folic acid was immobilized on the surface of try-AuNCs to ameliorate the specific affinity of AuNCs for tumors, and the near-infrared fluorescent FA-try-AuNCs were applied for in vivo cancer imaging of high folate receptor (FR) expressing Hela tumor. In vivo study of the dynamic behavior and targeting ability of FA-try-AuNCs probe to Hela tumor bearing mice and normal nude mice validated the high specific affinity of FA-try-AuNCs probe to FR positive tumors. The results show that the prepared try-AuNCs have great potential as multifunctional biomaterials for biosensing biomolecules with SPEET mode and in vivo cancer imaging with high targeting ability.

[Role of hemoglobin affinity to oxygen in adaptation to hypoxemia].

PubMed

Kwasiborski, Przemysław Jerzy; Kowalczyk, Paweł; Zieliński, Jakub; Przybylski, Jacek; Cwetsch, Andrzej

2010-04-01

One of the basic mechanisms of adapting to hypoxemia is a decrease in the affinity of hemoglobin for oxygen. This process occurs mainly due to the increased synthesis of 2,3-diphosphoglycerate (2,3-DPG) in the erythrocytes, as well as through the Bohr effect. Hemoglobin with decreased affinity for oxygen increases the oxygenation of tissues, because it gives up oxygen more easily during microcirculation. In foetal circulation, however, at a partial oxygen pressure (pO2) of 25 mmHg in the umbilical vein, the oxygen carrier is type F hemoglobin which has a high oxygen affinity. The commonly accepted role for hemoglobin F is limited to facilitating diffusion through the placenta. Is fetal life the only moment when haemoglobin F is useful? THE AIM OF STUDY was to create a mathematical model, which would answer the question at what conditions an increase, rather than a decrease, in haemoglobin oxygen affinity is of benefit to the body. Using the kinetics of dissociation of oxygen from hemoglobin described by the Hill equation as the basis for further discussion, we created a mathematical model describing the pO2 value in the microcirculatory system and its dependence on arterial blood pO2. The calculations were performed for hemoglobin with low oxygen affinity (adult type) and high-affinity hemoglobin (fetal type). The modelling took into account both physiological and pathological ranges of acid-base equilibrium and tissue oxygen extraction parameters. It was shown that for the physiological range of acid-base equilibrium and the resting level of tissue oxygen extraction parameters, with an arterial blood pO2 of 26.8 mmHg, the higher-affinity hemoglobin becomes the more effective oxygen carrier. It was also demonstrated that the arterial blood pO2, below which the high-affinity hemoglobin becomes the more effective carrier, is dependent on blood pH and the difference between the arterial and venous oxygen saturation levels. Simulations performed for the pathological

[Progresses in screening active compounds from herbal medicine by affinity chromatography].

PubMed

Feng, Ying-shu; Tong, Shan-shan; Xu, Xi-ming; Yu, Jiang-nan

2015-03-01

Affinity chromatography is a chromatographic method for separating molecules using the binding characteristics of the stationary phase with potential drug molecules. This method can be performed as a high throughput screening method and a chromatographic separation method to screen a variety of active drugs. This paper summarizes the history of affinity chromatography, screening technology of affinity chromatography, and application of affinity chromatography in screening bio-active compounds in herbal medicines, and then discusses its application prospects, in order to broaden applications of the affinity chromatography in drug screening.

Polydopamine Nanotubes as an Effective Fluorescent Quencher for Highly Sensitive and Selective Detection of Biomolecules Assisted with Exonuclease III Amplification.

PubMed

Fan, Daoqing; Zhu, Xiaoqing; Zhai, Qingfeng; Wang, Erkang; Dong, Shaojun

2016-09-20

In this work, the effective fluorescence quenching ability of polydopamine nanotubes (PDANTs) toward various fluorescent dyes was studied and further applied to fluorescent biosensing for the first time. The PDANTs could quench the fluorophores with different emission frequencies, aminomethylcoumarin acetate (AMCA), 6-carboxyfluorescein (FAM), 6-carboxytetramethylrhodamine (TAMRA), and Cy5. All the quenching efficiencies reached to more than 97%. Taking advantage of PDANTs' different affinities toward ssDNA and dsDNA and utilizing the complex of FAM-labeled ssDNA and PDANTs as a sensing platform, we achieved highly sensitive and selective detection of human immunodeficiency virus (HIV) DNA and adenosine triphosphate (ATP) assisted with Exonuclease III amplification. The limits of detection (LODs) of HIV DNA and ATP reached to 3.5 pM and 150 nM, respectively, which were all lower than that of previous nanoquenchers with Exo III amplification, and the platform also presented good applicability in biological samples. Fluorescent sensing applications of this nanotube enlightened other targets detection based upon it and enriched the building blocks of fluorescent sensing platforms. This polydopamine nanotube also possesses excellent biocompatibility and biodegradability, which is suitable for future drug delivery, cell imaging, and other biological applications.

Visualizing inducible nitric-oxide synthase in living cells with a heme-binding fluorescent inhibitor.

PubMed

Panda, Koustubh; Chawla-Sarkar, Mamta; Santos, Cecile; Koeck, Thomas; Erzurum, Serpil C; Parkinson, John F; Stuehr, Dennis J

2005-07-19

The study of nitric-oxide synthase (NOS) physiology is constrained by the lack of suitable probes to detect NOS in living cells or animals. Here, we characterized a fluorescent inducible NOS (iNOS) inhibitor called PIF (pyrimidine imidazole FITC) and examined its utility for microscopic imaging of iNOS in living cells. PIF binding to iNOS displayed high affinity, isoform selectivity, and heme specificity, and was essentially irreversible. PIF was used to successfully image iNOS expressed in RAW264.7 cells, HEK293T cells, human A549 epithelial cells, and freshly obtained human lung epithelium. PIF was used to estimate a half-life for iNOS of 1.8 h in HEK293T cells. Our work reveals that fluorescent probes like PIF will be valuable for studying iNOS cell biology and in understanding the pathophysiology of diseases that involve dysfunctional iNOS expression.

CdTe/CdSe quantum dots improve the binding affinities between α-amylase and polyphenols.

PubMed

Ni, Xiaoling

2012-03-01

People exposed to engineered nanomaterials have potential health risks associated. Human α-amylase is one of the key enzymes in the digestive system. There are few reports about the influence of quantum dots (QDs) on the digestive enzymes and their inhibition system. This work focused on the toxic effect of CdTe/CdSe QDs on the interactions between α-amylase and its natural inhibitors. Thirty-six dietary polyphenols, natural α-amylase inhibitors from food, were studied for their affinities for α-amylase in the absence and presence of CdTe/CdSe QDs by a fluorescence quenching method. The magnitudes of apparent binding constants of polyphenols for α-amylase were almost in the range of 10(5)-10(7) L mol(-1) in the presence of CdTe/CdSe QDs, which were higher than the magnitudes of apparent binding constants in the absence of CdTe/CdSe QDs (10(4)-10(6) L mol(-1)). CdTe/CdSe QDs obviously improved the affinities of dietary polyphenols for α-amylase up to 389.04 times. It is possible that the binding interaction between polyphenols and α-amylase in the presence of CdTe/CdSe QDs was mainly caused by electrostatic interactions. QDs significantly influence the digestive enzymes and their inhibition system. This journal is © The Royal Society of Chemistry 2012

Metal-enhanced fluorescence of dye-doped silica nano particles.

PubMed

Gunawardana, Kalani B; Green, Nathaniel S; Bumm, Lloyd A; Halterman, Ronald L

2015-03-01

Recent advancements in metal-enhanced fluorescence (MEF) suggest that it can be a promising tool for detecting molecules at very low concentrations when a fluorophore is fixed near the surface of metal nanoparticles. We report a simple method for aggregating multiple gold nanoparticles (GNPs) on Rhodamine B (RhB)-doped silica nanoparticles (SiNPs) utilizing dithiocarbamate (DTC) chemistry to produce MEF in solution. Dye was covalently incorporated into the growing silica framework via co-condensation of a 3-aminopropyltriethoxysilane (APTES) coupled RhB precursor using the Stöber method. Electron microscopy imaging revealed that these mainly non-spherical particles were relatively large (80 nm on average) and not well defined. Spherical core-shell particles were prepared by physisorbing a layer of RhB around a small spherical silica particle (13 nm) before condensing an outer layer of silica onto the surface. The core-shell method produced nanospheres (~30 nm) that were well defined and monodispersed. Both dye-doped SiNPs were functionalized with pendant amines that readily reacted with carbon disulfide (CS2) under basic conditions to produce DTC ligands that have exhibited a high affinity for gold surfaces. GNPs were produced via citrate reduction method and the resulting 13 nm gold nanospheres were then recoated with an ether-terminated alkanethiol to provide stability in ethanol. Fluorescent enhancement was observed when excess GNPs were added to DTC coated dye-doped SiNPs to form nanoparticle aggregates. Optimization of this system gave a fluorescence brightness enhancement of over 200 fold. Samples that gave fluorescence enhancement were characterized through Transmission Emission Micrograph (TEM) to reveal a pattern of multiple aggregation of GNPs on the dye-doped SiNPs.

Synthesis and evaluation of two NIR fluorescent cyclic RGD penta-peptides for targeting integrins

NASA Astrophysics Data System (ADS)

Ye, Yunpeng; Bloch, Sharon; Xu, Baogang; Achilefu, Samuel

2006-02-01

Interest in novel RGD peptides has been increasingly growing as the interactions between RGD peptides and integrins are the basis for a variety of cellular functions and medical applications such as modulation of cell adhesion, invasion, tumor angiogenesis, and metastasis. In particular, we have been interested in novel NIR fluorescent RGD peptides as potential optical contrast agents for in vivo tumor optical imaging. Therefore, two cyclic RGD penta-peptides conjugated with a NIR fluorescent carbocyanine (Cypate), i.e. lactam-based cyclo[RGDfK(Cypate)] (1) and disulfide-containing Cypate-cyclo(CRGDC)-NH II (2), were designed and synthesized. The competitive binding assay between the purified α vβ 3 integrin and the peptide ligands using 125I-echistatin as a tracer showed that 1 had a higher receptor binding affinity (IC 50~10 -7 M) than 2 (IC 50~10 -6 M). Furthermore, the internalization of 1 in A549 cells in vitro was less than 2, as revealed by fluorescence microscopy. These results suggest that both the lactam- and disulfide-based cyclic RGD penta-peptides should be further studied structurally and functionally to elucidate the advantages of each class of compounds.

Mathematical analysis of frontal affinity chromatography in particle and membrane configurations.

PubMed

Tejeda-Mansir, A; Montesinos, R M; Guzmán, R

2001-10-30

The scaleup and optimization of large-scale affinity-chromatographic operations in the recovery, separation and purification of biochemical components is of major industrial importance. The development of mathematical models to describe affinity-chromatographic processes, and the use of these models in computer programs to predict column performance is an engineering approach that can help to attain these bioprocess engineering tasks successfully. Most affinity-chromatographic separations are operated in the frontal mode, using fixed-bed columns. Purely diffusive and perfusion particles and membrane-based affinity chromatography are among the main commercially available technologies for these separations. For a particular application, a basic understanding of the main similarities and differences between particle and membrane frontal affinity chromatography and how these characteristics are reflected in the transport models is of fundamental relevance. This review presents the basic theoretical considerations used in the development of particle and membrane affinity chromatography models that can be applied in the design and operation of large-scale affinity separations in fixed-bed columns. A transport model for column affinity chromatography that considers column dispersion, particle internal convection, external film resistance, finite kinetic rate, plus macropore and micropore resistances is analyzed as a framework for exploring further the mathematical analysis. Such models provide a general realistic description of almost all practical systems. Specific mathematical models that take into account geometric considerations and transport effects have been developed for both particle and membrane affinity chromatography systems. Some of the most common simplified models, based on linear driving-force (LDF) and equilibrium assumptions, are emphasized. Analytical solutions of the corresponding simplified dimensionless affinity models are presented. Particular

Glowing locked nucleic acids: brightly fluorescent probes for detection of nucleic acids in cells.

PubMed

Østergaard, Michael E; Cheguru, Pallavi; Papasani, Madhusudhan R; Hill, Rodney A; Hrdlicka, Patrick J

2010-10-13

Fluorophore-modified oligonucleotides have found widespread use in genomics and enable detection of single-nucleotide polymorphisms, real-time monitoring of PCR, and imaging of mRNA in living cells. Hybridization probes modified with polarity-sensitive fluorophores and molecular beacons (MBs) are among the most popular approaches to produce hybridization-induced increases in fluorescence intensity for nucleic acid detection. In the present study, we demonstrate that the 2'-N-(pyren-1-yl)carbonyl-2'-amino locked nucleic acid (LNA) monomer X is a highly versatile building block for generation of efficient hybridization probes and quencher-free MBs. The hybridization and fluorescence properties of these Glowing LNA probes are efficiently modulated and optimized by changes in probe backbone chemistry and architecture. Correctly designed probes are shown to exhibit (a) high affinity toward RNA targets, (b) excellent mismatch discrimination, (c) high biostability, and (d) pronounced hybridization-induced increases in fluorescence intensity leading to formation of brightly fluorescent duplexes with unprecedented emission quantum yields (Φ(F) = 0.45-0.89) among pyrene-labeled oligonucleotides. Finally, specific binding between messenger RNA and multilabeled quencher-free MBs based on Glowing LNA monomers is demonstrated (a) using in vitro transcription assays and (b) by quantitative fluorometric assays and direct microscopic observation of probes bound to mRNA in its native form. These features render Glowing LNA as promising diagnostic probes for biomedical applications.

Weak affinity chromatography for evaluation of stereoisomers in early drug discovery.

PubMed

Duong-Thi, Minh-Dao; Bergström, Maria; Fex, Tomas; Svensson, Susanne; Ohlson, Sten; Isaksson, Roland

2013-07-01

In early drug discovery (e.g., in fragment screening), recognition of stereoisomeric structures is valuable and guides medicinal chemists to focus only on useful configurations. In this work, we concurrently screened mixtures of stereoisomers and estimated their affinities to a protein target (thrombin) using weak affinity chromatography-mass spectrometry (WAC-MS). Affinity determinations by WAC showed that minor changes in stereoisomeric configuration could have a major impact on affinity. The ability of WAC-MS to provide instant information about stereoselectivity and binding affinities directly from analyte mixtures is a great advantage in fragment library screening and drug lead development.

Evaluation of four affibody-based near-infrared fluorescent probes for optical imaging of epidermal growth factor receptor positive tumors.

PubMed

Qi, Shibo; Miao, Zheng; Liu, Hongguang; Xu, Yingding; Feng, Yaqing; Cheng, Zhen

2012-06-20

The epidermal growth factor receptor 1 (EGFR) has become an attractive target for cancer molecular imaging and therapy. An Affibody protein with strong binding affinity for EGFR, ZEGFR:1907, has been reported. We are interested in translating Affibody molecules to potential clinical optical imaging of EGFR positive cancers. In this study, four anti-EGFR Affibody based near-infrared (NIR) fluorescent probes were thus prepared, and their in vivo performance was evaluated in the mice bearing EGFR positive subcutaneous A431 tumors. The Affibody analogue, Ac-Cys-ZEGFR:1907, was synthesized using solid-phase peptide synthesis method. The purified small protein was then site-specifically conjugated with four NIR fluorescent dyes, Cy5.5-monomaleimide, Alex-Fluor-680-maleimide, SRfluor680-maleimide, or IRDye-800CW-maleimide, to produce four optical probes-Cy5.5-ZEGFR:1907, Alexa680-ZEGFR:1907, SR680-ZEGFR:1907, and 800CW-ZEGFR:1907. The EGFR binding property and specificity of the four NIR fluorescent Affibody probes were studied by fluorescence microscopy using high EGFR expressing A431 cells and low expressing MCF7 cells. The binding affinities of the probes (KD) to EGFR were further determined by flow cytometry. In vivo optical imaging of the four probes was performed in the mice bearing subcutaneous A431 tumors. The four NIR optical probes were prepared in high purity. In vitro cell imaging studies demonstrated that all of them could specifically bind to EGFR positive A431 cells while showing minimum uptake in low EGFR expressing MCF7 cells. Flow cytometry showed that Cy5.5-ZEGFR:1907 and Alexa680-ZEGFR:1907 possessed high binding affinity in low nanomolar range (43.6 ± 8.4 and 28.3 ± 4.9, respectively). In vivo optical imaging of the four probes revealed that they all showed fast tumor targeting ability and good tumor-to-normal tissue contrast as early as 0.5 h postinjection (p.i.). The tumor-to-normal tissue ratio reached a peak at 2 to 4 h p.i. by regional of

Synthesis of fluorescent dye-doped silica nanoparticles for target-cell-specific delivery and intracellular microRNA imaging.

PubMed

Li, Henan; Mu, Yawen; Qian, Shanshan; Lu, Jusheng; Wan, Yakun; Fu, Guodong; Liu, Songqin

2015-01-21

MicroRNA (miRNA) is found to be up-regulated in many kinds of cancer and therefore is classified as an oncomiR. Herein, we design a multifunctional fluorescent nanoprobe (FSiNP-AS/MB) with the AS1411 aptamer and a molecular beacon (MB) co-immobilized on the surface of the fluorescent dye-doped silica nanoparticles (FSiNPs) for target-cell-specific delivery and intracellular miRNA imaging. The FSiNPs were prepared by a facile reverse microemulsion method from tetraethoxysilane and silane derivatized coumarin that was previously synthesized by click chemistry. The as-prepared FSiNPs possess uniform size distribution, good optical stability and biocompatibility. In addition, there is a remarkable affinity interaction between the AS1411 aptamer and the nucleolin protein on the cancer cell surface. Thus, a target-cell-specific delivery system by the FSiNP-AS/MB is proposed for effectively transferring a MB into the cancer cells to recognize the target miRNA. Using miRNA-21 in MCF-7 cells (a human breast cancer cell line) as a model, the proposed multifunctional nanosystems not only allow target-cell-specific delivery with the binding affinity of AS1411, but also can track simultaneously the transfected cells and detect intracellular miRNA in situ. The proposed multifunctional nanosystems are a promising platform for a highly sensitive luminescent nonviral vector in biomedical and clinical research.

An in vivo imaging-based assay for detecting protein interactions over a wide range of binding affinities

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fowlkes, Jason Davidson; Owens, Elizabeth T; Standaert, Robert F

2009-01-01

Identifying and characterizing protein interactions are fundamental steps towards understanding and modeling biological networks. Methods that detect protein interactions in intact cells rather than buffered solutions are likely more relevant to natural systems since molecular crowding events in the cytosol can influence the diffusion and reactivity of individual proteins. One in vivo, imaging-based method relies on the co-localization of two proteins of interest fused to DivIVA, a cell division protein from Bacillus subtilis, and green fluorescent protein (GFP). We have modified this imaging-based assay to facilitate rapid cloning by constructing new vectors encoding N- and C-terminal DivIVA or GFP molecularmore » tag fusions based on site-specific recombination technology. The sensitivity of the assay was defined using a well-characterized protein interaction system involving the eukaryotic nuclear import receptor subunit, Importin (Imp ) and variant nuclear localization signals (NLS) representing a range of binding affinities. These data demonstrate that the modified co-localization assay is sensitive enough to detect protein interactions with Kd values that span over four orders of magnitude (1nM to 15 M). Lastly, this assay was used to confirm numerous protein interactions identified from mass spectrometry-based analyses of affinity isolates as part of an interactome mapping project in Rhodopseudomonas palustris« less

A carbon dot-based "off-on" fluorescent probe for highly selective and sensitive detection of phytic acid.

PubMed

Gao, Zhao; Wang, Libing; Su, Rongxin; Huang, Renliang; Qi, Wei; He, Zhimin

2015-08-15

We herein report a facile, one-step pyrolysis synthesis of photoluminescent carbon dots (CDs) using citric acid as the carbon source and lysine as the surface passivation reagent. The as-prepared CDs show narrow size distribution, excellent blue fluorescence and good photo-stability and water dispersivity. The fluorescence of the CDs was found to be effectively quenched by ferric (Fe(III)) ions with high selectivity via a photo-induced electron transfer (PET) process. Upon addition of phytic acid (PA) to the CDs/Fe(III) complex dispersion, the fluorescence of the CDs was significantly recovered, arising from the release of Fe(III) ions from the CDs/Fe(III) complex because PA has a higher affinity for Fe(III) ions compared to CDs. Furthermore, we developed an "off-on" fluorescence assay method for the detection of phytic acid using CDs/Fe(III) as a fluorescent probe. This probe enables the selective detection of PA with a linear range of 0.68-18.69 μM and a limit of detection (signal-to-noise ratio is 3) of 0.36 μM. The assay method demonstrates high selectivity, repeatability, stability and recovery ratio in the detection of the standard and real PA samples. We believe that the facile operation, low-cost, high sensitivity and selectivity render this CD-based "off-on" fluorescent probe an ideal sensing platform for the detection of PA. Copyright © 2015 Elsevier B.V. All rights reserved.

The high-affinity phosphodiesterase PdeH regulates development and aflatoxin biosynthesis in Aspergillus flavus.

PubMed

Yang, Kunlong; Liu, Yinghang; Liang, Linlin; Li, Zhenguo; Qin, Qiuping; Nie, Xinyi; Wang, Shihua

2017-04-01

Cyclic AMP signaling controls a range of physiological processes in response to extracellular stimuli in organisms. Among the signaling cascades, cAMP, as a second messenger, is orchestrated by adenylate cyclase (biosynthesis) and cAMP phosphodiesterases (PDEs) (hydrolysis). In this study, we investigated the function of the high-affinity (PdeH) and low-affinity (PdeL) cAMP phosphodiesterase from the carcinogenic aflatoxin producing fungus Aspergillus flavus, and found that instead of PdeL, inactivation of PdeH exhibited a reduction in conidiation and sclerotia formation. However, the ΔpdeL/ΔpdeH mutant exhibited an enhanced phenotype defects, a similar phenotype defects to wild-type strain treated with exogenous cAMP. The activation of PKA activity was inhibited in the ΔpdeH or ΔpdeL/ΔpdeH mutant, both of whom exhibited increasing AF production. Further analysis by qRT-PCR revealed that pdeH had a high transcriptional level compared to pdeL in wild-type strain, and affected pdeL transcription. Green fluorescent protein tagging at the C-terminus of PDEs showed that PdeH-GFP is broadly compartmentalized in the cytosol, while PdeL-GFP localized mainly to the nucleus. Overall, our results indicated that PdeH plays a major role, but has overlapping function with PdeL, in vegetative growth, development and AF biosynthesis in A. flavus. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

The major birch allergen, Bet v 1, shows affinity for a broad spectrum of physiological ligands.

PubMed

Mogensen, Jesper E; Wimmer, Reinhard; Larsen, Jørgen N; Spangfort, Michael D; Otzen, Daniel E

2002-06-28

Bet v 1 is a 17-kDa protein abundantly present in the pollen of the White birch tree and is the primary cause of birch pollen allergy in humans. Its three-dimensional structure is remarkable in that a solvent-accessible cavity traverses the core of the molecule. The biological function of Bet v 1 is unknown, although it is homologous to a family of pathogenesis-related proteins in plants. In this study we first show that Bet v 1 in the native state is able to bind the fluorescent probe 8-anilino-1-naphthalenesulfonic acid (ANS). ANS binds to Bet v 1 with 1:1 stoichiometry, and NMR data indicate that binding takes place in the cavity. Using an ANS displacement assay, we then identify a range of physiologically relevant ligands, including fatty acids, flavonoids, and cytokinins, which generally bind with low micromolar affinity. The ability of these ligands to displace ANS suggests that they also bind in the cavity, although the exact binding sites seem to vary among different ligands. The cytokinins, for example, seem to bind at a separate site close to ANS, because they increase the fluorescence of the ANS. Bet v 1 complex. Also, the fluorescent sterol dehydroergosterol binds to Bet v 1 as demonstrated by direct titrations. This study provides the first qualitative and quantitative data on the ligand binding properties of this important pollen allergen. Our findings indicate that ligand binding is important for the biological function of Bet v 1.

Bioengineering of Bacteria To Assemble Custom-Made Polyester Affinity Resins

PubMed Central

Hay, Iain D.; Du, Jinping; Burr, Natalie

2014-01-01

Proof of concept for the in vivo bacterial production of a polyester resin displaying various customizable affinity protein binding domains is provided. This was achieved by engineering various protein binding domains into a bacterial polyester-synthesizing enzyme. Affinity binding domains based on various structural folds and derived from molecular libraries were used to demonstrate the potential of this technique. Designed ankyrin repeat proteins (DARPins), engineered OB-fold domains (OBodies), and VHH domains from camelid antibodies (nanobodies) were employed. The respective resins were produced in a single bacterial fermentation step, and a simple purification protocol was developed. Purified resins were suitable for most lab-scale affinity chromatography purposes. All of the affinity domains tested produced polyester beads with specific affinity for the target protein. The binding capacity of these affinity resins ranged from 90 to 600 nmol of protein per wet gram of polyester affinity resin, enabling purification of a recombinant protein target from a complex bacterial cell lysate up to a purity level of 96% in one step. The polyester resin was efficiently produced by conventional lab-scale shake flask fermentation, resulting in bacteria accumulating up to 55% of their cellular dry weight as polyester. A further proof of concept demonstrating the practicality of this technique was obtained through the intracellular coproduction of a specific affinity resin and its target. This enables in vivo binding and purification of the coproduced “target protein.” Overall, this study provides evidence for the use of molecular engineering of polyester synthases toward the microbial production of specific bioseparation resins implementing previously selected binding domains. PMID:25344238

The increased binding affinity of curcumin with human serum albumin in the presence of rutin and baicalin: A potential for drug delivery system

NASA Astrophysics Data System (ADS)

Liu, Bing-Mi; Zhang, Jun; Hao, Ai-Jun; Xu, Liang; Wang, Dan; Ji, Hui; Sun, Shi-Jie; Chen, Bo-Qi; Liu, Bin

2016-02-01

The impacts of rutin and baicalin on the interaction of curcumin (CU) with human serum albumin (HSA) were investigated by fluorescence and circular dichroism (CD) spectroscopies under imitated physiological conditions. The results showed that the fluorescence quenching of HSA by CU was a simultaneous static and dynamic quenching process, irrespective of the presence or absence of flavonoids. The binding constants between CU and HSA in the absence and presence of rutin and baicalin were 2.268 × 105 M- 1, 3.062 × 105 M- 1, and 3.271 × 105 M- 1, indicating that the binding affinity was increased in the case of two flavonoids. Furthermore, the binding distance determined according to Förster's theory was decreased in the presence of flavonoids. Combined with the fact that flavonoids and CU have the same binding site (site I), it can be concluded that they may simultaneously bind in different regions in site I, and formed a ternary complex of flavonoid-HSA-CU. Meanwhile, the results of fluorescence quenching, CD and three-dimensional fluorescence spectra revealed that flavonoids further strengthened the microenvironmental and conformational changes of HSA induced by CU binding. Therefore, it is possible to develop a novel complex involving CU, flavonoid and HSA for CU delivery. The work may provide some valuable information in terms of improving the poor bioavailabiliy of CU.

In vivo gene transfer to dopamine neurons of rat substantia nigra via the high-affinity neurotensin receptor.

PubMed Central

Alvarez-Maya, I.; Navarro-Quiroga, I.; Meraz-Ríos, M. A.; Aceves, J.; Martinez-Fong, D.

2001-01-01

BACKGROUND: Recently, we synthesized a nonviral gene vector capable of transfecting cell lines taking advantage of neurotensin (NT) internalization. The vector is NT cross-linked with poly-L-lysine, to which a plasmid DNA was bound to form a complex (NT-polyplex). Nigral dopamine neurons are able to internalize NT, thus representing a target for gene transfer via NT-polyplex. This hypothesis was tested here using reporter genes encoding green fluorescent protein or chloramphenicol acetyl transferase. MATERIALS AND METHODS: NT-polyplex was injected into the substantia nigra. Double immunofluorescence labeling was used to reveal the cell type involved in the propidium iodide-labeled polyplex internalization and reporter gene expression. RESULTS: Polyplex internalization was observed within dopamine neurons but not within glial cells, and was prevented by both hypertonic sucrose solution and SR-48692, a selective nonpeptide antagonist of NT receptors. Reporter gene expression was observed in dopamine neurons from 48 hr up to 15 days after NT-polyplex injection, and was prevented by SR-48692. However, no expression was seen when the NT-polyplex was injected into the ansiform lobule of the cerebellum, which contains low- but not high-affinity NT receptors. Neither internalization nor expression was observed in cultured glial cells, despite the NT-polyplex binding to those cells that was prevented by levocabastine, a low-affinity NT receptor antagonist. CONCLUSIONS: These results suggest that high-affinity NT receptors mediate the uptake of NT-polyplex with the subsequent reporter gene expression in vivo. NT polyfection may be used to transfer genes of physiologic interest to nigrostriatal dopamine neurons, and to produce transgenic animal models of dopamine-related diseases. PMID:11471555

Multi-Affinity for Growing Rough Interfaces of Bacterial Colonies

NASA Astrophysics Data System (ADS)

Kobayashi, N.; Ozawa, T.; Saito, K.; Yamazaki, Y.; Matsuyama, T.; Matsushita, M.

We have examined whether rough interfaces of bacterial colonies are multi-affine. We have used the bacterial species called textit{Bacillus subtilis}, which has been found to exhibit a variety of colony patterns when varying both the concentration of nutrient and solidity of agar medium. Consequently, we have found that the colony interface on a nutrient-rich, solid agar medium is multi-affine. On the other hand, the colony interface on a nutrient-rich, semi-solid agar medium is self-affine.

Characterization of self-affinity in the global regime

NASA Astrophysics Data System (ADS)

Neimark, Alexander V.

1994-11-01

Methods for characterization of self-affine surfaces and measurements of their roughness exponents H are developed. It is shown that for smoothed surfaces, which underwent particular coarse graining or averaging of the small-scale fluctuations, the excess surface area Sex and the mean square root radius of curvature ac are related by two distinct asymptotic power laws if ac is well below or well above a certain crossover scale acr. In the local regime of self-affinity, when ac<affinity, when ac>>acr, Sex~(ac/acr)-2(1-H)/(2-H). The former scaling relationship is consistent with the well known definition of local fractal dimensions dloc=dtop+1-H. The latter scaling relationship offers alternatives for characterization of self-affinity over large scales by means of excess dimensions defined as dex=dtop+2(1-H)/(2-H) and can be used for determination of roughness exponents from the measurements provided in the global regime. The thermodynamic method of fractal analysis, proposed earlier for self-similar surfaces (A.V. Neimark, Pis'ma Zh. Eksp. Teor. Fiz. 51, 535 (1990) [JETP Lett. 51, 607 (1990)]; Physica A 191, 258 (1992)), is extended for self-affine surfaces for determination of fractal dimensions and roughness exponents from adsorption and capillary experimental data.

A novel fluorescent aptasensor for the highly sensitive and selective detection of cardiac troponin I based on a graphene oxide platform.

PubMed

Liu, Dongkui; Lu, Xing; Yang, Yiwen; Zhai, Yunyun; Zhang, Jian; Li, Lei

2018-05-04

Acute myocardial infarction (AMI) is one of the leading risks to global health. Thus, the rapid, accurate early diagnosis of AMI is highly critical. Human cardiac troponin I (cTnI) has been regarded as a golden biomarker for AMI due to its excellent selectivity. In this work, a novel fluorescent aptasensor based on a graphene oxide (GO) platform was developed for the highly sensitive and selective detection of cTnI. GO binds to the fluorescent anti-cTnI aptamer and quenches its fluorescence. In the presence of cTnI, the fluorescent anti-cTnI aptamer leaves the surface of GO, combines with cTnI because of the powerful affinity of the fluorescent anti-cTnI aptamer and cTnI, and then restores the fluorescence of the fluorescent anti-cTnI aptamer. Fluorescence-enhanced detection is highly sensitive and selective to cTnI. The method exhibited good analytical performance with a reasonable dynamic linearity at the concentration range of 0.10-6.0 ng/mL and a low detection limit of 0.07 ng/mL (S/N = 3). The fluorescent aptasensor also exhibited high selectivity toward cTnI compared with other interference proteins. The proposed method may be a potentially useful tool for cTnI determination in human serum. Graphical abstract A novel fluorescent aptasensor for the highly sensitive and selective detection of cardiac troponin I based on a graphene oxide platform.

Hydride affinities of cumulated, isolated, and conjugated dienes in acetonitrile.

PubMed

Zhu, Xiao-Qing; Liang, Hao; Zhu, Yan; Cheng, Jin-Pei

2008-11-07

The hydride affinities (defined as the enthalpy changes in this work) of 15 polarized dienes [five phenyl sulfone substituted allenes (1a), the corresponding five isolated dienes (1b), and the corresponding five conjugated dienes (1c)] in acetonitrile solution were determined by titration calorimetry for the first time. The results display that the hydride affinity scales of the 15 dienes in acetonitrile range from -71.6 to -73.9 kcal/mol for 1a, from -46.2 to -49.7 kcal/mol for 1b, and from -45.0 to -46.5 kcal/mol for 1c, which indicates that the hydride-obtaining abilities of the cumulated dienes (1a) are not only much larger than those of the corresponding conjugated dienes (1c) but also much larger than those of the corresponding isolated dienes (1b). The hydrogen affinities of the 15 dienes as well as the hydrogen affinities and the proton affinities of the radical anions of the dienes (1(-*)) in acetonitrile were also evaluated by using relative thermodynamic cycles according to Hess's law. The results show that (i) the hydrogen affinities of the neutral dienes 1 cover a range from -44.5 to -45.6 kcal/mol for 1a, from -20.4 to -21.4 kcal/mol for 1b, and from -17.3 to -18.5 kcal/mol for 1c; (ii) the hydrogen affinities of the radical anions of the dienes (1(-*)) in acetonitrile cover a range from -40.6 to -47.2 kcal/mol for 1a(-*), from -21.6 to -29.6 kcal/mol for 1b(-*), and from -10.0 to -15.4 kcal/mol for 1c(-*); (iii) the proton affinities of the 15 1a(-*) in acetonitrile cover a range from -97.0 to -100.6 kcal/mol for 1a(-*), from -77.8 to -83.4 kcal/mol for 1b(-*), and from -66.2 to -68.9 kcal/mol for 1c(-*). The main reasons for the great difference between the cumulated dienes and the corresponding isolated and conjugated dienes in the hydride affinity, hydrogen affinity, and proton affinity have been examined. It is evident that these experimental results should be quite valuable to facilitate the elucidation of the origins of the especially high

Comparison of energy interaction parameters for the complexation of Pr(III) with glutathione reduced (GSH) in absence and presence of Zn(II) in aqueous and aquated organic solvents using 4f?4f transition spectra as PROBE

NASA Astrophysics Data System (ADS)

Singh, Th. David; Sumitra, Ch.; Yaiphaba, N.; Devi, H. Debecca; Devi, M. Indira; Singh, N. Rajmuhon

2005-04-01

The coordination chemistry of glutathione reduced (GSH) is of great importance as it acts as excellent model system for the binding of metal ions. The GSH complexation with metal ions is involved in the toxicology of different metal ions. Its coordination behaviour for soft metal ions and hard metal ions is found different because of the structure of GSH and its different potential binding sites. In our work we have studied two chemically dissimilar metal ions viz. Pr(III), which prefer hard donor site like carboxylic groups and Zn(II) the soft metal ion which prefer peptide-NH and sulphydryl groups. The absorption difference and comparative absorption spectroscopy involving 4f-4f transitions of the heterobimetallic Complexation of GSH with Pr(III) and Zn(II) has been explored in aqueous and aquated organic solvents. The variation in the energy parameters like Slater-Condon ( F K), Racah ( E K) and Lande ( ξ4f), Nephelauxetic parameter ( β) and bonding parameter ( b1/2) are computed to explain the nature of complexation.

Zn(II), Cd(II) and Hg(I) complexes of cinnamic acid: FT-IR, FT-Raman, 1H and 13C NMR studies

NASA Astrophysics Data System (ADS)

Kalinowska, M.; Świsłocka, R.; Lewandowski, W.

2011-05-01

The effect of zinc, cadmium(II) and mercury(I) ions on the electronic structure of cinnamic acid (phenylacrylic acid) was studied. In this research many miscellaneous analytical methods, which complement one another, were used: infrared (FT-IR), Raman (FT-Raman), nuclear magnetic resonance ( 1H, 13C NMR) and quantum mechanical calculations. The spectroscopic studies provide some knowledge on the distribution of the electronic charge in molecule, the delocalization energy of π-electrons and the reactivity of metal complexes. In the series of Zn(II) → Cd(II) → Hg(I) cinnamates: (1) systematic shifts of several bands in the experimental and theoretical IR and Raman spectra and (2) regular chemical shifts for protons 1H and 13C nuclei were observed.

Mapping multivalency and differential affinities within large intrinsically disordered protein complexes with segmental motion analysis.

PubMed

Milles, Sigrid; Lemke, Edward A

2014-07-07

Intrinsically disordered proteins (IDPs) can bind to multiple interaction partners. Numerous binding regions in the IDP that act in concert through complex cooperative effects facilitate such interactions, but complicate studying IDP complexes. To address this challenge we developed a combined fluorescence correlation and time-resolved polarization spectroscopy approach to study the binding properties of the IDP nucleoporin153 (Nup153) to nuclear transport receptors (NTRs). The detection of segmental backbone mobility of Nup153 within the unperturbed complex provided a readout of local, region-specific binding properties that are usually masked in measurements of the whole IDP. The binding affinities of functionally and structurally diverse NTRs to distinct regions of Nup153 can differ by orders of magnitudes-a result with implications for the diversity of transport routes in nucleocytoplasmic transport. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Higher Nucleoporin-Importinβ Affinity at the Nuclear Basket Increases Nucleocytoplasmic Import

PubMed Central

Azimi, Mohammad; Mofrad, Mohammad R. K.

2013-01-01

Several in vitro studies have shown the presence of an affinity gradient in nuclear pore complex proteins for the import receptor Importinβ, at least partially contributing to nucleocytoplasmic transport, while others have historically argued against the presence of such a gradient. Nonetheless, the existence of an affinity gradient has remained an uncharacterized contributing factor. To shed light on the affinity gradient theory and better characterize how the existence of such an affinity gradient between the nuclear pore and the import receptor may influence the nucleocytoplasmic traffic, we have developed a general-purpose agent based modeling (ABM) framework that features a new method for relating rate constants to molecular binding and unbinding probabilities, and used our ABM approach to quantify the effects of a wide range of forward and reverse nucleoporin-Importinβ affinity gradients. Our results indicate that transport through the nuclear pore complex is maximized with an effective macroscopic affinity gradient of 2000 µM, 200 µM and 10 µM in the cytoplasmic, central channel and nuclear basket respectively. The transport rate at this gradient is approximately 10% higher than the transport rate for a comparable pore lacking any affinity gradient, which has a peak transport rate when all nucleoporins have an affinity of 200 µM for Importinβ. Furthermore, this optimal ratio of affinity gradients is representative of the ratio of affinities reported for the yeast nuclear pore complex – suggesting that the affinity gradient seen in vitro is highly optimized. PMID:24282617

An affinity-directed protein missile system for targeted proteolysis.

PubMed

Fulcher, Luke J; Macartney, Thomas; Bozatzi, Polyxeni; Hornberger, Annika; Rojas-Fernandez, Alejandro; Sapkota, Gopal P

2016-10-01

The von Hippel-Lindau (VHL) protein serves to recruit the hypoxia-inducible factor alpha (HIF1α) protein under normoxia to the CUL2 E3 ubiquitin ligase for its ubiquitylation and degradation through the proteasome. In this report, we modify VHL to engineer an affinity-directed protein missile (AdPROM) system to direct specific endogenous target proteins for proteolysis in mammalian cells. The proteolytic AdPROM construct harbours a cameloid anti-green fluorescence protein (aGFP) nanobody that is fused to VHL for either constitutive or tetracycline-inducible expression. For target proteins, we exploit CRISPR/Cas9 to rapidly generate human kidney HEK293 and U2OS osteosarcoma homozygous knock-in cells harbouring GFP tags at the VPS34 (vacuolar protein sorting 34) and protein associated with SMAD1 (PAWS1, aka FAM83G) loci, respectively. Using these cells, we demonstrate that the expression of the VHL-aGFP AdPROM system results in near-complete degradation of the endogenous GFP-VPS34 and PAWS1-GFP proteins through the proteasome. Additionally, we show that Tet-inducible destruction of GFP-VPS34 results in the degradation of its associated partner, UVRAG, and reduction in levels of cellular phosphatidylinositol 3-phosphate. © 2016 The Authors.

An affinity-directed protein missile system for targeted proteolysis

PubMed Central

Fulcher, Luke J.; Macartney, Thomas; Bozatzi, Polyxeni; Hornberger, Annika; Rojas-Fernandez, Alejandro

2016-01-01

The von Hippel–Lindau (VHL) protein serves to recruit the hypoxia-inducible factor alpha (HIF1α) protein under normoxia to the CUL2 E3 ubiquitin ligase for its ubiquitylation and degradation through the proteasome. In this report, we modify VHL to engineer an affinity-directed protein missile (AdPROM) system to direct specific endogenous target proteins for proteolysis in mammalian cells. The proteolytic AdPROM construct harbours a cameloid anti-green fluorescence protein (aGFP) nanobody that is fused to VHL for either constitutive or tetracycline-inducible expression. For target proteins, we exploit CRISPR/Cas9 to rapidly generate human kidney HEK293 and U2OS osteosarcoma homozygous knock-in cells harbouring GFP tags at the VPS34 (vacuolar protein sorting 34) and protein associated with SMAD1 (PAWS1, aka FAM83G) loci, respectively. Using these cells, we demonstrate that the expression of the VHL-aGFP AdPROM system results in near-complete degradation of the endogenous GFP-VPS34 and PAWS1-GFP proteins through the proteasome. Additionally, we show that Tet-inducible destruction of GFP-VPS34 results in the degradation of its associated partner, UVRAG, and reduction in levels of cellular phosphatidylinositol 3-phosphate. PMID:27784791

Methyl cation affinities of neutral and anionic maingroup-element hydrides: trends across the periodic table and correlation with proton affinities.

PubMed

Mulder, R Joshua; Guerra, Célia Fonseca; Bickelhaupt, F Matthias

2010-07-22

We have computed the methyl cation affinities in the gas phase of archetypal anionic and neutral bases across the periodic table using ZORA-relativistic density functional theory (DFT) at BP86/QZ4P//BP86/TZ2P. The main purpose of this work is to provide the methyl cation affinities (and corresponding entropies) at 298 K of all anionic (XH(n-1)(-)) and neutral bases (XH(n)) constituted by maingroup-element hydrides of groups 14-17 and the noble gases (i.e., group 18) along the periods 2-6. The cation affinity of the bases decreases from H(+) to CH(3)(+). To understand this trend, we have carried out quantitative bond energy decomposition analyses (EDA). Quantitative correlations are established between the MCA and PA values.

Asymptotic Representations of Quantum Affine Superalgebras

NASA Astrophysics Data System (ADS)

Zhang, Huafeng

2017-08-01

We study representations of the quantum affine superalgebra associated with a general linear Lie superalgebra. In the spirit of Hernandez-Jimbo, we construct inductive systems of Kirillov-Reshetikhin modules based on a cyclicity result that we established previously on tensor products of these modules, and realize their inductive limits as modules over its Borel subalgebra, the so-called q-Yangian. A new generic asymptotic limit of the same inductive systems is proposed, resulting in modules over the full quantum affine superalgebra. We derive generalized Baxter's relations in the sense of Frenkel-Hernandez for representations of the full quantum group.

Relationships between chemical structure and affinity for acetylcholine receptors

PubMed Central

Abramson, F. B.; Barlow, R. B.; Mustafa, M. G.; Stephenson, R. P.

1969-01-01

1. Series of analogues of acetylcholine have been prepared in which the acetyl group was replaced by phenylacetyl, cyclohexylacetyl, diphenylacetyl, dicyclohexylacetyl, (±)-phenylcyclohexylacetyl, benziloyl and (±)-phenylcyclohexylhydroxyacetyl groups and the trimethylammonium group was replaced by Me2EtN+, MeEt2N+, Et3N+, [Formula: see text] Further series were prepared in which the acetoxyethyl group was replaced by ethoxyethyl, phenylethoxyethyl, cyclohexylethoxyethyl, diphenylethoxyethyl, and dicyclohexylethoxyethyl groups, and by n-pentyl, 5-phenylpentyl, 5-cyclohexylpentyl and 5:5-diphenylpentyl groups. 2. The ethoxyethyl and n-pentyl series contain some compounds which are agonists or partial agonists when tested on the isolated guinea-pig ileum, but all the other compounds are antagonists. 3. The affinity of the compounds for the postganglionic (“muscarinesensitive”) acetylcholine receptors has been measured in conditions in which the antagonists have been shown to be acting competitively. There were considerable differences between their affinities, the most active (log K, 9·8) having one million times the affinity of the least active (log K, 3·7). 4. The changes in affinity as the onium group was modified were not entirely independent of changes in the rest of the molecule. Increasing the size of the onium group, as judged from conductivity measurements on simpler onium salts, increased affinity in the series containing one large group (phenyl or cyclohexyl) but, in the series with two large groups, affinity declined when the size was increased beyond -+NMeEt2. 5. In general, the effects of changes in the rest of the molecule on affinity were bigger than the effects of changes in the onium group and there were bigger interactions. Affinity was increased to a greater extent by introducing one phenyl and one cyclohexyl group together than by introducing either two phenyl or two cyclohexyl groups; the increment was greater than the separate

Identification of polypeptides with selective affinity to intact mouse cerebellar granule neurons from a random peptide-presenting phage library.

PubMed

Hou, Sheng T; Dove, Mike; Anderson, Erica; Zhang, Jiangbing; MacKenzie, C Roger

2004-09-30

Targeting of postmitotic neurons selectively for gene delivery poses a challenge. One way to achieve such a selective targeting is to link the gene delivery vector with small ligand-binding polypeptides which have selective affinity to intact neurons. In order to identify such novel neuron selective polypeptides, we screened a phage-display library displaying random 12-mer polypeptides and subtractively bio-panned for clones having selectivity towards cultured mouse cerebellar granule neurons. The selected phage clones were amplified and sequenced. Affinities of these clones to neurons were determined by the visible presence or absence of fluorescence of phage particles as detected by immunocytochemistry using an antibody to M-13 phage. This affinity was further qualified by how much phage was bound, and where in or on the cell it tended to accumulate. The selectivity of binding to neurons was determined by the negative binding of these clones to several cultured non-neuronal cells, including, primary glial cells, NT2 cells, human embryonic kidney 293 cells, neuroblastoma cells, and mouse 3T3 cells. Among the 46 clones that we have sequenced and characterized, four clones appeared to have excellent selectivity in binding to neurons. Homology comparison of these polypeptides revealed that three of them contained a consensus D(E)-W(F)-I(N)-D-W motif. This motif was also present in the Bdm1 gene product which was predominantly expressed in postnatal brains. Further characterizations of these polypeptides are required to reveal the utilities of these peptides to function as an effective linker to facilitate gene transfer selectively to neurons.

Red cell 2,3-diphosphoglycerate and oxygen affinity.

PubMed

MacDonald, R

1977-06-01

The ease with which haemoglobin releases oxygen to the tissues is controlled by erythrocytic 2,3-diphosphoglycerate (2,3-DPG) such that an increase in the concentration of 2,3-DPG decreases oxygen affinity and vice versa. This review article describes the synthesis and breakdown of 2,3-DPG in the Embden-Meyerof pathway in red cells and briefly explains the molecular basis for its effect on oxygen affinity. Interaction of the effects of pH, Pco2, temperature and 2,3-DPG on the oxyhaemoglobin dissociation curve are discussed. The role of 2,3-DPG in the intraerythrocytic adaptation to various types of hypoxaemia is described. The increased oxygen affinity of blood stored in acid-citrate-dextrose (ACD) solution has been shown to be due to the decrease in the concentration of 2,3-DPG which occurs during storage. Methods of maintaining the concentration of 2,3-DPG in stored blood are described. The clinical implication of transfusion of elderly people, anaemic or pregnant patients with ACD stored blood to anaesthetically and surgically acceptable haemoglobin concentrations are discussed. Hypophosphataemia in association with parenteral feeding reduces 2,3-DPG concentration and so increases oxygen affinity. Since post-operative use of intravenous fluids such as dextrose or dextrose/saline also lead to hypophosphataemia, the addition of inorganic phosphorus to routine post-operative intravenous fluid may be advisable. Disorders of acid-base balance effect oxygen affinity not only by the direct effect of pH on the oxyhaemoglobin dissociation curve but by its control of 2,3-DPG metabolism. Management of acid-base disorders and pre-operative aklalinization of patients with sickle cell disease whould take account of this. It is known that anaesthesia alters the position of the oxyhaemoglobin dissociation curve, but it is thought that this is independent of any effects which anaesthetic agents may have on 2,3-DPG concentration. In vitro manipulation of 2,3-DPG concentration

Affinity Electrophoresis Using Ligands Attached To Polymers

NASA Technical Reports Server (NTRS)

Van Alstine, James M.; Snyder, Robert S.; Harris, J. M.; Brooks, D. E.

1990-01-01

In new technique, reduction of electrophoretic mobilities by addition of polyethylene glycol to ligands increases electrophoretic separabilities. In immuno-affinity electrophoresis, modification of ligands extends specificity of electrophoretic separation to particles having surface electric-charge structures otherwise making them electrophoretically inseparable. Modification of antibodies by polyethylene glycol greatly reduces ability to aggregate while enhancing ability to affect electrophoretic mobilities of cells. In hydrophobic-affinity electrophoresis, addition of polyethylene glycol reduces tendency toward aggregation of cells or macromolecules.

Employing linear tetranuclear [Zn4(COO)4(OH)2] clusters as building subunits to construct a new Zn(II) coordination polymer with tunable luminescent properties

NASA Astrophysics Data System (ADS)

Li, Wu-Wu; Zhang, Zun-Ting

2016-02-01

A new Zn(II) coordination polymer, [Zn2(btc) (biimpy) (OH)]n (1 H3btc = 1,3,5-benzenetricarboxylic acid, biimpy = 2,6-bis(1-imdazoly)pyridine) has been successfully synthesized and characterized by elemental analysis, powder single crystal X-ray diffraction analyses. Compound 1 features a 3D framework employing linear tetranuclear [Zn4(COO)4(OH)2] cluster as building subunits. Topological analysis reveals it represents a (3,10)-connected structural topology by viewing btc3-, linear tetranuclear clusters and biimpy as 3-connected nodes, 10-connected nodes, linear linkers, respectively. Moreover, the thermal stability and luminescent property of compound 1 have been well investigated.

Syntheses, structures and photoluminescence properties of three M(II)-coordination polymers (M dbnd Zn(II), Mn(II)) based on a pyridine N-oxide bridging ligand

NASA Astrophysics Data System (ADS)

Ren, Xiu-Hui; Wang, Peng; Cheng, Jun-Yan; Dong, Yu-Bin

2018-06-01

Three M(II)-coordination polymers (M dbnd Zn(II), Mn(II)) were synthesized based on a pyridine N-oxide bridging ligand 3,5-bis(4-carboxylphenyl)-pyridine N-oxide (L1). Compounds 1-3 all have novel complicated structures. Compound 1 (Zn(L1)2(H2O)2) and 2 (Zn2(L1)2(H2O)2) are two single crystals obtained in "one pot" and 1 features 1D double chains motif and 2 features 3D network structure. Compound 3 shows 3D network structure with triangular tunnels. The thermogravimetric analyses and photoluminescence properties were also used to investigate the title compounds.

A novel fluorescent DNA sensor for ultrasensitive detection of Helicobacter pylori.

PubMed

Liu, Ziping; Su, Xingguang

2017-01-15

In this work, a novel fluorescent DNA sensor for ultrasensitive detection of Helicobacter pylori (H. pylori) DNA was developed. This strategy took advantage of DNA hybridization between single-stranded DNA (ssDNA, which had been designed as an aptamer specific for H. pylori DNA) and the complementary target H. pylori DNA, and the feature that ssDNA bound to graphene oxide (GO) with significantly higher affinity than double-stranded DNA (dsDNA). ssDNA were firstly covalent conjugated with CuInS 2 quantum dots (QDs) by reaction between the carboxy group of QDs and amino group modified ssDNA, forming ssDNA-QDs genosensor. In the absence of the complementary target H. pylori DNA, GO could adsorb ssDNA-QDs DNA sensor and efficiently quench the fluorescence of ssDNA-QDs. While the complementary target H. pylori DNA was introduced, the ssDNA-QDs preferentially bound with the H. pylori DNA. The formation of dsDNA would alter the conformation of ssDNA and disturb the interaction between ssDNA and GO. Thus, the dsDNA-QDs/GO system exhibited a stronger fluorescence emission than that of the ssDNA-QDs/GO system. Under the optimized conditions, a linear correlation was established between the fluorescence intensity ratio I/I 0 and the concentration of H. pylori DNA in the range of 1.25-875pmolL -1 with a detection limit of 0.46pmolL -1 . The proposed method was applied to the determination of H. pylori DNA sequence in milk samples with satisfactory results. Copyright © 2016 Elsevier B.V. All rights reserved.

Camelid VHH affinity ligands enable separation of closely related biopharmaceuticals

PubMed Central

Pabst, Timothy M.; Wendeler, Michaela; Wang, Xiangyang; Bezemer, Sandra; Hermans, Pim

2016-01-01

Abstract Interest in new and diverse classes of molecules such as recombinant toxins, enzymes, and blood factors continues to grow for use a biotherapeutics. Compared to monoclonal antibodies, these novel drugs typically lack a commercially available affinity chromatography option, which leads to greater process complexity, longer development timelines, and poor platformability. To date, for both monoclonal antibodies and novel molecules, affinity chromatography has been mostly reserved for separation of process‐related impurities such as host cell proteins and DNA. Reports of affinity purification of closely related product variants and modified forms are much rarer. In this work we describe custom affinity chromatography development using camelid VHH antibody fragments as "tunable" immunoaffinity ligands for separation of product‐related impurities. One example demonstrates high selectivity for a recombinant immunotoxin where no binding was observed for an undesired deamidated species. Also discussed is affinity purification of a coagulation factor through specific recognition of the gamma‐carboxylglutamic acid domain. PMID:27677057

Exploring Girls' Science Affinities Through an Informal Science Education Program

NASA Astrophysics Data System (ADS)

Todd, Brandy; Zvoch, Keith

2017-10-01

This study examines science interests, efficacy, attitudes, and identity—referred to as affinities, in the context of an informal science outreach program for girls. A mixed methods design was used to explore girls' science affinities before, during, and after participation in a cohort-based summer science camp. Multivariate analysis of survey data revealed that girls' science affinities varied as a function of the joint relationship between family background and number of years in the program, with girls from more affluent families predicted to increase affinities over time and girls from lower income families to experience initial gains in affinities that diminish over time. Qualitative examination of girls' perspectives on gender and science efficacy, attitudes toward science, and elements of science identities revealed a complex interplay of gendered stereotypes of science and girls' personal desires to prove themselves knowledgeable and competent scientists. Implications for the best practice in fostering science engagement and identities in middle school-aged girls are discussed.

Selective and Sensitive Detection of Cyanide Based on the Displacement Strategy Using a Water-Soluble Fluorescent Probe.

PubMed

La, Ming; Hao, Yuanqiang; Wang, Zhaoyang; Han, Guo-Cheng; Qu, Lingbo

2016-01-01

A water-soluble fluorescent probe (C-GGH) was used for the highly sensitive and selective detection of cyanide (CN(-)) in aqueous media based on the displacement strategy. Due to the presence of the recognition unit GGH (Gly-Gly-His), the probe C-GGH can coordinate with Cu(2+) and consequently display ON-OFF type fluorescence response. Furthermore, the in situ formed nonfluorescent C-GGH-Cu(2+) complex can act as an effective OFF-ON type fluorescent probe for sensing CN(-) anion. Due to the strong binding affinity of CN(-) to Cu(2+), CN(-) can extract Cu(2+) from C-GGH-Cu(2+) complex, leading to the release of C-GGH and the recovery of fluorescent emission of the system. The probe C-GGH-Cu(2+) allowed detection of CN(-) in aqueous solution with a LOD (limit of detection) of 0.017 μmol/L which is much lower than the maximum contaminant level (1.9 μmol/L) for CN(-) in drinking water set by the WHO (World Health Organization). The probe also displayed excellent specificity for CN(-) towards other anions, including F(-), Cl(-), Br(-), I(-), SCN(-), PO4 (3-), N3 (-), NO3 (-), AcO(-), SO4 (2-), and CO3 (2-).

Selective and Sensitive Detection of Cyanide Based on the Displacement Strategy Using a Water-Soluble Fluorescent Probe

PubMed Central

La, Ming; Hao, Yuanqiang; Wang, Zhaoyang; Han, Guo-Cheng; Qu, Lingbo

2016-01-01

A water-soluble fluorescent probe (C-GGH) was used for the highly sensitive and selective detection of cyanide (CN−) in aqueous media based on the displacement strategy. Due to the presence of the recognition unit GGH (Gly-Gly-His), the probe C-GGH can coordinate with Cu2+ and consequently display ON-OFF type fluorescence response. Furthermore, the in situ formed nonfluorescent C-GGH-Cu2+ complex can act as an effective OFF-ON type fluorescent probe for sensing CN− anion. Due to the strong binding affinity of CN− to Cu2+, CN− can extract Cu2+ from C-GGH-Cu2+ complex, leading to the release of C-GGH and the recovery of fluorescent emission of the system. The probe C-GGH-Cu2+ allowed detection of CN− in aqueous solution with a LOD (limit of detection) of 0.017 μmol/L which is much lower than the maximum contaminant level (1.9 μmol/L) for CN− in drinking water set by the WHO (World Health Organization). The probe also displayed excellent specificity for CN− towards other anions, including F−, Cl−, Br−, I−, SCN−, PO4 3−, N3 −, NO3 −, AcO−, SO4 2−, and CO3 2−. PMID:26881185

Reversible and Dynamic Fluorescence Imaging of Cellular Redox Self-Regulation Using Fast-Responsive Near-Infrared Ge-Pyronines.

PubMed

Nie, Hailiang; Jing, Jing; Tian, Yong; Yang, Wen; Zhang, Rubo; Zhang, Xiaoling

2016-04-13

Cellular self-regulation of reactive oxygen species (ROS) stress via glutathione (GSH) antioxidant repair plays a crucial role in maintaining redox balance, which affects various physiological and pathological pathways. In this work, we developed a simple yet effective strategy for reversible, dynamic, and real-time fluorescence imaging of ROS stress and GSH repair, based on novel Ge-pyronine dyes (GePs). Unlike the current O-pyronine (OP) dye, the fluorescence of GePs can be quenched in GSH reduction and then greatly restored by ROS (e.g., ClO(-), ONOO(-), and HO(•)) oxidation because of their unique affinity toward thiols. The "on-off" and "off-on" fluorescence switch can complete in 10 and 20 s, respectively, and exhibit excellent reversibility in vitro and in cells. GePs also show excitation in the long wavelength from the deep-red to near-infrared (NIR) (621-662 nm) region, high fluorescence quantum yield (Φ(fl) = 0.32-0.44) in aqueous media, and excellent cell permeability. Our results demonstrated that GePs can be used for real-time monitoring of the reversible and dynamic interconversion between ROS oxidation and GSH reduction in living cells. GePs might be a useful tool for investigating various redox-related physiological and pathological pathways.

Coordination of N,O-donor appended Schiff base ligand (H2L1) towards Zinc(II) in presence of pseudohalides: Syntheses, crystal structures, photoluminescence, antimicrobial activities and Hirshfeld surfaces

NASA Astrophysics Data System (ADS)

Majumdar, Dhrubajyoti; Biswas, Jayanta Kumar; Mondal, Monojit; Surendra Babu, M. S.; Metre, Ramesh K.; Das, Sourav; Bankura, Kalipada; Mishra, Dipankar

2018-03-01

A series of dinuclear Zn(II) complexes [Zn2 (L1) (CH3OH)2(SCN) (OAc)](1), [Zn2 (L1) (CH3OH)2(N3)2](2) and [Zn2 (L1) (Cl)2(CH3OH)]·CH3OH (3) have been synthesized by the reaction of compartmental Schiff base ligand (H2L1) [N,N‧-Bis(3-ethoxysalicylidenimino)-1,3-diaminopropane] with Zn(OAc)2·2H2O in presence of coligand like KSCN, NaN3 and NaCl respectively. X-ray diffraction analysis revealed that all the complexes are neutral and possess a 4-membered Zn2 (μ2-O)2 ring fastened by the unified coordination action of a doubly deprotonated ligand. In addition, solid state structure of the complexes display extensive intermolecular interaction which has been supported theoretically by Hirshfeld surface analysis with 2D Fingerprint plots. The synthesized Zn(II) metal complexes observed enhancement of luminescence emission compared to the parent Schiff base due to emanating ligand based intraligand (π→π∗) fluorescence. Additionally, Zn(II) metal complexes exhibited considerable antimicrobial potency against some important Gram +ve and Gram -ve bacteria.

Europium-Labeled Synthetic C3a Protein as a Novel Fluorescent Probe for Human Complement C3a Receptor.

PubMed

Dantas de Araujo, Aline; Wu, Chongyang; Wu, Kai-Chen; Reid, Robert C; Durek, Thomas; Lim, Junxian; Fairlie, David P

2017-06-21

Measuring ligand affinity for a G protein-coupled receptor is often a crucial step in drug discovery. It has been traditionally determined by binding putative new ligands in competition with native ligand labeled with a radioisotope of finite lifetime. Competing instead with a lanthanide-based fluorescent ligand is more attractive due to greater longevity, stability, and safety. Here, we have chemically synthesized the 77 residue human C3a protein and conjugated its N-terminus to europium diethylenetriaminepentaacetate to produce a novel fluorescent protein (Eu-DTPA-hC3a). Time-resolved fluorescence analysis has demonstrated that Eu-DTPA-hC3a binds selectively to its cognate G protein-coupled receptor C3aR with full agonist activity and similar potency and selectivity as native C3a in inducing calcium mobilization and phosphorylation of extracellular signal-regulated kinases in HEK293 cells that stably expressed C3aR. Time-resolved fluorescence analysis for saturation and competitive binding gave a dissociation constant (K d ) of 8.7 ± 1.4 nM for Eu-DTPA-hC3a and binding affinities for hC3a (pK i of 8.6 ± 0.2 and K i of 2.5 nM) and C3aR ligands TR16 (pK i of 6.8 ± 0.1 and K i of 138 nM), BR103 (pK i of 6.7 ± 0.1 and K i of 185 nM), BR111 (pK i of 6.3 ± 0.2 and K i of 544 nM) and SB290157 (pK i of 6.3 ± 0.1 and K i of 517 nM) via displacement of Eu-DTPA-hC3a from hC3aR. The macromolecular conjugate Eu-DTPA-hC3a is a novel nonradioactive probe suitable for studying ligand-C3aR interactions with potential value in accelerating drug development for human C3aR in physiology and disease.

Motion planning in velocity affine mechanical systems

NASA Astrophysics Data System (ADS)

Jakubiak, Janusz; Tchoń, Krzysztof; Magiera, Władysław

2010-09-01

We address the motion planning problem in specific mechanical systems whose linear and angular velocities depend affinely on control. The configuration space of these systems encompasses the rotation group, and the motion planning involves the system orientation. Derivation of the motion planning algorithm for velocity affine systems has been inspired by the continuation method. Performance of this algorithm is illustrated with examples of the kinematics of a serial nonholonomic manipulator, the plate-ball kinematics and the attitude control of a rigid body.

Purification of phage display-modified bacteriophage T4 by affinity chromatography

PubMed Central

2011-01-01

Background Affinity chromatography is one of the most efficient protein purification strategies. This technique comprises a one-step procedure with a purification level in the order of several thousand-fold, adaptable for various proteins, differentiated in their size, shape, charge, and other properties. The aim of this work was to verify the possibility of applying affinity chromatography in bacteriophage purification, with the perspective of therapeutic purposes. T4 is a large, icosahedral phage that may serve as an efficient display platform for foreign peptides or proteins. Here we propose a new method of T4 phage purification by affinity chromatography after its modification with affinity tags (GST and Histag) by in vivo phage display. As any permanent introduction of extraneous DNA into a phage genome is strongly unfavourable for medical purposes, integration of foreign motifs with the phage genome was not applied. The phage was propagated in bacteria expressing fusions of the phage protein Hoc with affinity tags from bacterial plasmids, independently from the phage expression system. Results Elution profiles of phages modified with the specific affinity motifs (compared to non-specific phages) document their binding to the affinity resins and effective elution with standard competitive agents. Non-specific binding was also observed, but was 102-105 times weaker than the specific one. GST-modified bacteriophages were also effectively released from glutathione Sepharose by proteolytic cleavage. The possibility of proteolytic release was designed at the stage of expression vector construction. Decrease in LPS content in phage preparations was dependent on the washing intensity; intensive washing resulted in preparations of 11-40 EU/ml. Conclusions Affinity tags can be successfully incorporated into the T4 phage capsid by the in vivo phage display technique and they strongly elevate bacteriophage affinity to a specific resin. Affinity chromatography can be

Measurements of the talus in the assessment of population affinity.

PubMed

Bidmos, Mubarak A; Dayal, Manisha R; Adegboye, Oyelola A

2018-06-01

As part of their routine work, forensic anthropologists are expected to report population affinity as part of the biological profile of an individual. The skull is the most widely used bone for the estimation of population affinity but it is not always present in a forensic case. Thus, other bones that preserve well have been shown to give a good indication of either the sex or population affinity of an individual. In this study, the potential of measurements of the talus was investigated for the purpose of estimating population affinity in South Africans. Nine measurements from two hundred and twenty tali of South African Africans (SAA) and South African Whites (SAW) from the Raymond A. Dart Collection of Human Skeletons were used. Direct and step-wise discriminant function and logistic regression analyses were carried out using SPSS and SAS. Talar length was the best single variable for discriminating between these two groups for males while in females the head height was the best single predictor. Average accuracies for correct population affinity classification using logistic regression analysis were higher than those obtained from discriminant function analysis. This study was the first of its type to employ discriminant function analyses and logistic regression analyses to estimate the population affinity of an individual from the talus. Thus these equations can now be used by South African anthropologists when estimating the population affinity of dismembered or damaged or incomplete skeletal remains of SAA and SAW. Copyright © 2018 Elsevier B.V. All rights reserved.

Excited state electron affinity calculations for aluminum

NASA Astrophysics Data System (ADS)

Hussein, Adnan Yousif

2017-08-01

Excited states of negative aluminum ion are reviewed, and calculations of electron affinities of the states (3s^23p^2)^1D and (3s3p^3){^5}{S}° relative to the (3s^23p)^2P° and (3s3p^2)^4P respectively of the neutral aluminum atom are reported in the framework of nonrelativistic configuration interaction (CI) method. A priori selected CI (SCI) with truncation energy error (Bunge in J Chem Phys 125:014107, 2006) and CI by parts (Bunge and Carbó-Dorca in J Chem Phys 125:014108, 2006) are used to approximate the valence nonrelativistic energy. Systematic studies of convergence of electron affinity with respect to the CI excitation level are reported. The calculated value of the electron affinity for ^1D state is 78.675(3) meV. Detailed Calculations on the ^5S°c state reveals that is 1216.8166(3) meV below the ^4P state.

Supramolecular Affinity Chromatography for Methylation-Targeted Proteomics.

PubMed

Garnett, Graham A E; Starke, Melissa J; Shaurya, Alok; Li, Janessa; Hof, Fraser

2016-04-05

Proteome-wide studies of post-translationally methylated species using mass spectrometry are complicated by high sample diversity, competition for ionization among peptides, and mass redundancies. Antibody-based enrichment has powered methylation proteomics until now, but the reliability, pan-specificity, polyclonal nature, and stability of the available pan-specific antibodies are problematic and do not provide a standard, reliable platform for investigators. We have invented an anionic supramolecular host that can form host-guest complexes selectively with methyllysine-containing peptides and used it to create a methylysine-affinity column. The column resolves peptides on the basis of methylation-a feat impossible with a comparable commercial cation-exchange column. A proteolyzed nuclear extract was separated on the methyl-affinity column prior to standard proteomics analysis. This experiment demonstrates that such chemical methyl-affinity columns are capable of enriching and improving the analysis of methyllysine residues from complex protein mixtures. We discuss the importance of this advance in the context of biomolecule-driven enrichment methods.

Metal ion release from metallothioneins: proteolysis as an alternative to oxidation.

PubMed

Peroza, Estevão A; dos Santos Cabral, Augusto; Wan, Xiaoqiong; Freisinger, Eva

2013-09-01

Metallothioneins (MTs) are among others involved in the cellular regulation of essential Zn(II) and Cu(I) ions. However, the high binding affinity of these proteins requires additional factors to promote metal ion release under physiological conditions. The mechanisms and efficiencies of these processes leave many open questions. We report here a comprehensive analysis of the Zn(II)-release properties of various MTs with special focus on members of the four main subfamilies of plant MTs. Zn(II) competition experiments with the metal ion chelator 4-(2-pyridylazo)resorcinol (PAR) in the presence of the cellular redox pair glutathione (GSH)/glutathione disulfide (GSSG) show that plant MTs from the subfamilies MT1, MT2, and MT3 are remarkably more affected by oxidative stress than those from the Ec subfamily and the well-characterized human MT2 form. In addition, we evaluated proteolytic digestion with trypsin and proteinase K as an alternative mechanism for selective promotion of metal ion release from MTs. Also here the observed percentage of liberated metal ions depends strongly on the MT form evaluated. Closer evaluation of the data additionally allowed deducing the thermodynamic and kinetic properties of the Zn(II) release processes. The Cu(I)-form of chickpea MT2 was used to exemplify that both oxidation and proteolysis are also effective ways to increase the transfer of copper ions to other molecules. Zn(II) release experiments with the individual metal-binding domains of Ec-1 from wheat grain reveal distinct differences from the full-length protein. This triggers the question about the roles of the long cysteine-free peptide stretches typical for plant MTs.

Quinoline Fluorescent Probes for Zinc - from Diagnostic to Therapeutic Molecules in Treating Neurodegenerative Diseases.

PubMed

Czaplinska, Barbara; Spaczynska, Ewelina; Musiol, Robert

2018-01-01

Fluorescent compounds had gained strong attention due to their wide and appealing applications. Microscopic techniques and visualization are good examples among others. Introduction of fluorescent dyes into microbiology opens the possibility to observe tissues, organisms or organelle with exceptional sensitivity and resolution. Probes for detection of biologically relevant metals as zinc, iron or copper seems to be particularly important for drug design and pharmaceutical sciences. Quinoline derivatives are well known for their good metal affinity and wide spectrum of biological activity. In this regard, molecular sensors built on this scaffold may be useful not only as analytical but also as therapeutic agents. In the present review, application of quinoline moiety in designing of novel fluorescent probes for zinc is presented and discussed. Zinc cations are relevant for vast majority of processes and recently attract a great deal of attention for their role in neurodegenerative diseases. Compounds interacting with Zn2+ may be used for early diagnosis of such disorders, for example the Alzheimer disease. Quinoline-based zinc probes may exert some beneficial role in organism acting as theranostic agents. First preliminary drugs for Alzheimer therapy that are based on quinoline moiety are good example of this trend. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

PRINCIPLES OF AFFINITY-BASED BIOSENSORS

EPA Science Inventory

Despite the amount of resources that have been invested by national and international academic, government, and commercial sectors to develop affinity-based biosensor products, little obvious success has been realized through commercialization of these devices for specific applic...

Protein purification by aminosquarylium cyanine dye-affinity chromatography.

PubMed

Silva, M S; Graça, V C; Reis, L V; Santos, P F; Almeida, P; Queiroz, J A; Sousa, F

2013-12-01

The most selective purification method for proteins and other biomolecules is affinity chromatography. This method is based on the unique biological-based specificity of the biomolecule-ligand interaction and commonly uses biological ligands. However, these ligands may present some drawbacks, mainly because of their cost and lability. Dye-affinity chromatography overcomes the limitations of biological ligands and is widely used owing to the low cost of synthetic dyes and to their resistance to biological and chemical degradation. In this work, immobilized aminosquarylium cyanine dyes are used in order to exploit affinity interactions with standard proteins such as lysozyme, α-chymotrypsin and trypsin. These studies evaluate the affinity interactions occurring between the immobilized ligand and the different proteins, as a reflection of the sum of several molecular interactions, namely ionic, hydrophobic and van der Waals, spread throughout the structure, in a defined spatial manner. The results show the possibility of using an aminosquarylium cyanine dye bearing a N-hexyl pendant chain, with a ligand density of 1.8 × 10(-2) mmol of dye/g of chromatographic support, to isolate lysozyme, α-chymotrypsin and trypsin from a mixture. The application of a decreasing ammonium sulfate gradient resulted in the recovery of lysozyme in the flowthrough. On the other hand, α-chymotrypsin and trypsin were retained, involving different interactions with the ligand. In conclusion, this study demonstrates the potential applicability of ligands such as aminosquarylium cyanine dyes for the separation and purification of proteins by affinity chromatography. Copyright © 2013 John Wiley & Sons, Ltd.

Shark Attack: high affinity binding proteins derived from shark vNAR domains by stepwise in vitro affinity maturation.

PubMed

Zielonka, Stefan; Weber, Niklas; Becker, Stefan; Doerner, Achim; Christmann, Andreas; Christmann, Christine; Uth, Christina; Fritz, Janine; Schäfer, Elena; Steinmann, Björn; Empting, Martin; Ockelmann, Pia; Lierz, Michael; Kolmar, Harald

2014-12-10

A novel method for stepwise in vitro affinity maturation of antigen-specific shark vNAR domains is described that exclusively relies on semi-synthetic repertoires derived from non-immunized sharks. Target-specific molecules were selected from a CDR3-randomized bamboo shark (Chiloscyllium plagiosum) vNAR library using yeast surface display as platform technology. Various antigen-binding vNAR domains were easily isolated by screening against several therapeutically relevant antigens, including the epithelial cell adhesion molecule (EpCAM), the Ephrin type-A receptor 2 (EphA2), and the human serine protease HTRA1. Affinity maturation was demonstrated for EpCAM and HTRA1 by diversifying CDR1 of target-enriched populations which allowed for the rapid selection of nanomolar binders. EpCAM-specific vNAR molecules were produced as soluble proteins and more extensively characterized via thermal shift assays and biolayer interferometry. Essentially, we demonstrate that high-affinity binders can be generated in vitro without largely compromising the desirable high thermostability of the vNAR scaffold. Copyright © 2014 Elsevier B.V. All rights reserved.

Supercritical angle fluorescence as a tool to study the interaction between lipid bilayer and peptides

NASA Astrophysics Data System (ADS)

Dubois, Valentin; Serrano, Diana; Seeger, Stefan

2017-06-01

The understanding of processes occurring at the interface between two media are of prior importance in various fields of research, from material sciences to biology. A custom-made microscope objective based on the supercritical angle technique was developed in our group, allowing to probe these interfacial events by carrying out surface-sensitive and low invasive spectroscopy of aqueous samples. A biological example of particular interest is the comprehension of neurodegenerative diseases which seem caused by the interaction of specific peptides with the membrane of the neurons. Taking advantage of our optical setup, we used supercritical angle fluorescence spectroscopy to specifically monitor the interaction between a supported lipid bilayer (SLB) and the Amyloid β peptide, notably responsible of the Alzheimer disease. Different forms of the peptide (40 and 42 amino acids composition) were tested and the interfacial fluorescence measured to get information about the lipid integrity and mobility. The adsorption of the peptide was also characterized in terms of kinetic and affinity.

Determination of residual fluoroquinolones in honey by liquid chromatography using metal chelate affinity chromatography.

PubMed

Yatsukawa, Yoh-Ichi; Ito, Hironobu; Matsuda, Takahiro; Nakamura, Munetomo; Watai, Masatoshi; Fujita, Kazuhiro

2011-01-01

A new analytical method for the simultaneous determination of seven fluoroquinolones, namely, norfloxacin, ciprofloxacin, danofloxacin, enrofloxacin, orbifloxacin, sarafloxacin, and difloxacin, especially in dark-colored honey, has been developed. Fluoroquinolone antibiotics were extracted from samples with MacIlvaine buffer solution (pH 4.0) containing EDTA disodium salt dihydrate. The extracts were treated with both a polymeric cartridge and a metal chelate affinity column preloaded with ferric ion (Fe3+). LC separation with fluorescence detection was performed at 40 degrees C using an Inertsil ODS-4 analytical column (150 x 4.6 mm, 3 microm). The mobile phase was composed of 20 mM/L citrate buffer solution (pH 3.1)-acetonitrile mixture (70 + 30, v/v) containing 1 mM/L sodium dodecyl sulfate. Lomefloxacin was used as an internal standard. The developed method was validated according to the criteria of European Commission Decision 2002/657/EC. Decision limits and detection capabilities were below 2.9 and 4.4 microg/kg, respectively.

Bidirectional Elastic Image Registration Using B-Spline Affine Transformation

PubMed Central

Gu, Suicheng; Meng, Xin; Sciurba, Frank C.; Wang, Chen; Kaminski, Naftali; Pu, Jiantao

2014-01-01

A registration scheme termed as B-spline affine transformation (BSAT) is presented in this study to elastically align two images. We define an affine transformation instead of the traditional translation at each control point. Mathematically, BSAT is a generalized form of the affine transformation and the traditional B-Spline transformation (BST). In order to improve the performance of the iterative closest point (ICP) method in registering two homologous shapes but with large deformation, a bi-directional instead of the traditional unidirectional objective / cost function is proposed. In implementation, the objective function is formulated as a sparse linear equation problem, and a sub-division strategy is used to achieve a reasonable efficiency in registration. The performance of the developed scheme was assessed using both two-dimensional (2D) synthesized dataset and three-dimensional (3D) volumetric computed tomography (CT) data. Our experiments showed that the proposed B-spline affine model could obtain reasonable registration accuracy. PMID:24530210

Increased hemoglobin O2 affinity protects during acute hypoxia

PubMed Central

Yalcin, Ozlem

2012-01-01

Acclimatization to hypoxia requires time to complete the adaptation mechanisms that influence oxygen (O2) transport and O2 utilization. Although decreasing hemoglobin (Hb) O2 affinity would favor the release of O2 to the tissues, increasing Hb O2 affinity would augment arterial O2 saturation during hypoxia. This study was designed to test the hypothesis that pharmacologically increasing the Hb O2 affinity will augment O2 transport during severe hypoxia (10 and 5% inspired O2) compared with normal Hb O2 affinity. RBC Hb O2 affinity was increased by infusion of 20 mg/kg of 5-hydroxymethyl-2-furfural (5HMF). Control animals received only the vehicle. The effects of increasing Hb O2 affinity were studied in the hamster window chamber model, in terms of systemic and microvascular hemodynamics and partial pressures of O2 (Po2). Pimonidazole binding to hypoxic areas of mice heart and brain was also studied. 5HMF decreased the Po2 at which the Hb is 50% saturated with O2 by 12.6 mmHg. During 10 and 5% O2 hypoxia, 5HMF increased arterial blood O2 saturation by 35 and 48% from the vehicle group, respectively. During 5% O2 hypoxia, blood pressure and heart rate were 58 and 30% higher for 5HMF compared with the vehicle. In addition, 5HMF preserved microvascular blood flow, whereas blood flow decreased to 40% of baseline in the vehicle group. Consequently, perivascular Po2 was three times higher in the 5HMF group compared with the control group at 5% O2 hypoxia. 5HMF also reduced heart and brain hypoxic areas in mice. Therefore, increased Hb O2 affinity resulted in hemodynamics and oxygenation benefits during severe hypoxia. This acute acclimatization process may have implications in survival during severe environmental hypoxia when logistic constraints prevent chronic acclimatization. PMID:22636677

Interplay between binding affinity and kinetics in protein-protein interactions.

PubMed

Cao, Huaiqing; Huang, Yongqi; Liu, Zhirong

2016-07-01

To clarify the interplay between the binding affinity and kinetics of protein-protein interactions, and the possible role of intrinsically disordered proteins in such interactions, molecular simulations were carried out on 20 protein complexes. With bias potential and reweighting techniques, the free energy profiles were obtained under physiological affinities, which showed that the bound-state valley is deep with a barrier height of 12 - 33 RT. From the dependence of the affinity on interface interactions, the entropic contribution to the binding affinity is approximated to be proportional to the interface area. The extracted dissociation rates based on the Arrhenius law correlate reasonably well with the experimental values (Pearson correlation coefficient R = 0.79). For each protein complex, a linear free energy relationship between binding affinity and the dissociation rate was confirmed, but the distribution of the slopes for intrinsically disordered proteins showed no essential difference with that observed for ordered proteins. A comparison with protein folding was also performed. Proteins 2016; 84:920-933. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Pb, Cu, and Zn distributions at humic acid-coated metal-oxide surfaces

NASA Astrophysics Data System (ADS)

Wang, Yingge; Michel, F. Marc; Choi, Yongseong; Eng, Peter J.; Levard, Clement; Siebner, Hagar; Gu, Baohua; Bargar, John R.; Brown, Gordon E.

2016-09-01

Mineral surfaces are often coated by natural organic matter (NOM), which has a major influence on metal-ion sorption and sequestration because of the abundance of binding sites in such coatings and the changes they cause in local nanoscale environments. The effects of NOM coatings on mineral surfaces are, however, still poorly understood at the molecular level due to the complexity of these systems. In this study, we have applied long-period X-ray standing wave-fluorescence yield (LP-XSW-FY) spectroscopy to measure the partitioning of naturally present Cu(II) (0.0226%), Zn(II) (0.009%), and Pb(II) (∼0.0004%) between Elliott Soil Humic Acid (ESHA) coatings and three model single-crystal metal-oxide substrates: α-Al2O3 (0 0 0 1), α-Al2O3 (1 -1 0 2), and α-Fe2O3 (0 0 0 1). The competitive sorption effects among these metal ions for binding sites in the ESHA coatings and on the metal-oxide surfaces were investigated as a function of reaction time, calcium content, and solution pH. Pb(II) ions present in the ESHA coatings were found to redistribute to reactive α-Al2O3 (1 -1 0 2) and α-Fe2O3 (0 0 0 1) surfaces after 3 h of reaction (pH = 6.0, [Ca(II)] = 2 mM). Pb(II) partitioning onto these reactive metal-oxide surfaces increased with increasing reaction time (up to 7 d). In addition, the partitioning of Cu(II) and Zn(II) from the ESHA coating to the α-Fe2O3 (0 0 0 1) substrate increased slightly with reaction time (2.4% and 3.7% for Cu(II) and Zn(II), respectively, after 3 h and 6.4% and 7.7% for Cu(II) and Zn(II), respectively, after 72 h of reaction time). However, no changes in the partitioning of Cu(II) and Zn(II) onto the α-Al2O3 (1 -1 0 2) surface were observed with increasing reaction time, suggesting that these ions strongly complex with functional groups in the ESHA coatings. Similar results were obtained for Cu(II) and Zn(II) on the ESHA-coated α-Al2O3 (1 -1 0 2) surfaces in samples without the addition of calcium. However, the amounts of Pb

Synthesis of highly water-soluble fluorescent conjugated glycopoly(p-phenylene)s for lectin and Escherichia coli.

PubMed

Xue, Cuihua; Jog, Sonali P; Murthy, Pushpalatha; Liu, Haiying

2006-09-01

Two facile, convenient, and versatile synthetic approaches are used to covalently attach carbohydrate residues to conjugated poly(p-phenylene)s (PPPs) for highly water-soluble PPPs bearing alpha-mannopyranosyl and beta-glucopyranosyl pendants (polymers A and B), which highly fluoresce in phosphate buffer (pH 7.0). The post-polymerization functionalization approach is to treat bromo-bearing PPP (polymer 1) with 1-thiolethyl-alpha-D-mannose tetraacetate or 1-thiol-beta-D-glucose tetraacetate in THF solution in the presence of K(2)CO(3) at room temperature through formation of thioether bridges, affording polymer 2a or 2b. The prepolymerization functionalization approach is to polymerize a well-defined sugar-carrying monomer, affording polymer 2a. Polymers 2a and 2b were deacetylated under Zemplén conditions in methanol and methylene chloride containing sodium methoxide, affording polymers A and B, respectively. The multivalent display of carbohydrates on the fluorescent conjugated glycopolymer overcomes the characteristic low binding affinity of the individual carbohydrates to their receptor proteins. Titration of concanavalin A (Con A) to alpha-mannose-bearing polymer A resulted in significant fluorescent quenching of the polymer with Stern-Volmer quenching constant of 4.5 x 10(7). Incubation of polymer A with Escherichia coli (E. coli) lead to formation of fluorescently stained bacterial clusters. Beta-glucose-bearing polymer B displayed no response to Con A and E. coli.

G-protein signaling leverages subunit-dependent membrane affinity to differentially control βγ translocation to intracellular membranes.

PubMed

O'Neill, Patrick R; Karunarathne, W K Ajith; Kalyanaraman, Vani; Silvius, John R; Gautam, N

2012-12-18

Activation of G-protein heterotrimers by receptors at the plasma membrane stimulates βγ-complex dissociation from the α-subunit and translocation to internal membranes. This intermembrane movement of lipid-modified proteins is a fundamental but poorly understood feature of cell signaling. The differential translocation of G-protein βγ-subunit types provides a valuable experimental model to examine the movement of signaling proteins between membranes in a living cell. We used live cell imaging, mathematical modeling, and in vitro measurements of lipidated fluorescent peptide dissociation from vesicles to determine the mechanistic basis of the intermembrane movement and identify the interactions responsible for differential translocation kinetics in this family of evolutionarily conserved proteins. We found that the reversible translocation is mediated by the limited affinity of the βγ-subunits for membranes. The differential kinetics of the βγ-subunit types are determined by variations among a set of basic and hydrophobic residues in the γ-subunit types. G-protein signaling thus leverages the wide variation in membrane dissociation rates among different γ-subunit types to differentially control βγ-translocation kinetics in response to receptor activation. The conservation of primary structures of γ-subunits across mammalian species suggests that there can be evolutionary selection for primary structures that confer specific membrane-binding affinities and consequent rates of intermembrane movement.

Binding affinities of anti-acetylcholine receptor autoantibodies in myasthenia gravis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bray, J.J.; Drachman, D.B.

1982-01-01

Antibodies directed against acetylcholine (ACh) receptors are present in the sera of nearly 90% of patients with myasthenia gravis (MG), and are involved in the pathogenesis of this autoimmune disease. However, the antibody titers measured by the standard radioimmunoassay correspond poorly with the clinical severity of the disease. To determine whether this disparity could be accounted for by differences in the binding affinities of anti-ACh receptor antibodies in different patients, we have measured the binding affinities of these autoantibodies in 15 sera from MG patients. The affinity constants (K/sub o/), as determined by Scatchard analysis, were all in the rangemore » of 10/sup 10/ M/sup -1/, comparable to the highest values reported in immunized animals. The affinity constants were truly representative of the population of autoantibodies detected by the radioimmunoassay, as shown by the remarkable linearity of the Scatchard plots (r/sup 2/>0.90) and the close correlation between the antibody titers determined by extrapolation of the Scatchard plots and by saturation analysis (r = 0.99; p < 0.001). There was only a 6-fold variation in affinity constants measured in this series of patients despite widely differing antibody titers and severity of the disease. Factors other than the titer and affinity of anti-ACh receptor antibodies may correlate better with the clinical manifestations of MG.« less

Regulatory mechanisms of hemoglobin oxygen affinity in acidosis and alkalosis

PubMed Central

Bellingham, A. J.; Detter, J. C.; Lenfant, C.

1971-01-01

The recent reports of the effect of 2,3-diphosphoglycerate (2,3-DPG) on hemoglobin affinity for oxygen suggested that this substance may play a role in man's adaptation to acidosis and alkalosis. A study of the effect of induced acidosis and alkalosis on the oxyhemoglobin dissociation curve of normal man was therefore carried out, and the mechanisms involved in the physiological regulation of hemoglobin oxygen affinity examined. In acute changes of plasma pH there was no alteration in red cell 2,3-DPG content. However, there were changes in hemoglobin oxygen affinity and these correlated with changes in mean corpuscular hemoglobin concentration (MCHC). With maintained acidosis and alkalosis, red cell 2,3-DPG content was altered and correlated with the changes in hemoglobin oxygen affinity. Both of these mechanisms shift the hemoglobin oxygen dissociation curve opposite to the direct pH (Bohr) effect, and providing the rate of pH change is neither too rapid nor too large, they counteract the direct pH effect and the in vivo hemoglobin oxygen affinity remains unchanged. It is also shown that approximately 35% of the change in hemoglobin oxygen affinity resulting from an alteration in red cell 2,3-DPG, is explained by effect of 2,3-DPG on the red cell pH. PMID:5545127

An improved affine projection algorithm for active noise cancellation

NASA Astrophysics Data System (ADS)

Zhang, Congyan; Wang, Mingjiang; Han, Yufei; Sun, Yunzhuo

2017-08-01

Affine projection algorithm is a signal reuse algorithm, and it has a good convergence rate compared to other traditional adaptive filtering algorithm. There are two factors that affect the performance of the algorithm, which are step factor and the projection length. In the paper, we propose a new variable step size affine projection algorithm (VSS-APA). It dynamically changes the step size according to certain rules, so that it can get smaller steady-state error and faster convergence speed. Simulation results can prove that its performance is superior to the traditional affine projection algorithm and in the active noise control (ANC) applications, the new algorithm can get very good results.

Development of an aptamer-conjugated fluorescent nanoprobe for MMP2

NASA Astrophysics Data System (ADS)

Han, Myoung-Eun; Baek, Sungmin; Kim, Hyun-Jung; Lee, Jung Hwan; Ryu, Sung-Ho; Oh, Sae-Ock

2014-03-01

Matrix metalloproteinase 2 (MMP2) plays critical roles in various diseases, such as atherosclerosis and cancer, and has been suggested to contribute to the instability of atherosclerotic plaque. To visualize MMP2 in pathologic tissues, we developed an aptamer targeting MMP2 protein by performing eight rounds of modified DNA systematic evolution of ligands by exponential enrichment (SELEX). The aptamer showed high affinity for MMP2 ( K d = 5.59 nM), precipitated MMP2, and detected MMP2 protein in pathological tissues such as atherosclerotic plaque and gastric cancer tissues. Furthermore, a MMP2 aptamer-conjugated fluorescent nanoprobe successfully visualized atherosclerotic plaques in apolipoprotein E (ApoE) knockout mice. These results suggest that the devised MMP2 aptamer could be useful for the development of various diagnostic tools.

Characterizing informative sequence descriptors and predicting binding affinities of heterodimeric protein complexes.

PubMed

Srinivasulu, Yerukala Sathipati; Wang, Jyun-Rong; Hsu, Kai-Ti; Tsai, Ming-Ju; Charoenkwan, Phasit; Huang, Wen-Lin; Huang, Hui-Ling; Ho, Shinn-Ying

2015-01-01

Protein-protein interactions (PPIs) are involved in various biological processes, and underlying mechanism of the interactions plays a crucial role in therapeutics and protein engineering. Most machine learning approaches have been developed for predicting the binding affinity of protein-protein complexes based on structure and functional information. This work aims to predict the binding affinity of heterodimeric protein complexes from sequences only. This work proposes a support vector machine (SVM) based binding affinity classifier, called SVM-BAC, to classify heterodimeric protein complexes based on the prediction of their binding affinity. SVM-BAC identified 14 of 580 sequence descriptors (physicochemical, energetic and conformational properties of the 20 amino acids) to classify 216 heterodimeric protein complexes into low and high binding affinity. SVM-BAC yielded the training accuracy, sensitivity, specificity, AUC and test accuracy of 85.80%, 0.89, 0.83, 0.86 and 83.33%, respectively, better than existing machine learning algorithms. The 14 features and support vector regression were further used to estimate the binding affinities (Pkd) of 200 heterodimeric protein complexes. Prediction performance of a Jackknife test was the correlation coefficient of 0.34 and mean absolute error of 1.4. We further analyze three informative physicochemical properties according to their contribution to prediction performance. Results reveal that the following properties are effective in predicting the binding affinity of heterodimeric protein complexes: apparent partition energy based on buried molar fractions, relations between chemical structure and biological activity in principal component analysis IV, and normalized frequency of beta turn. The proposed sequence-based prediction method SVM-BAC uses an optimal feature selection method to identify 14 informative features to classify and predict binding affinity of heterodimeric protein complexes. The characterization

Syntheses, structures, photoluminescence of four dicarboxylate-controlled Zn(II) coordination complexes incorporating flexible 1-(4-pyridylmethyl)-benzimidazole ligand

NASA Astrophysics Data System (ADS)

Hao, Hong-Jun; Du, Ming-Yue; Wang, Dan-Feng; Sun, Cheng-Jie; Wang, Zhan-Hui; Huang, Rong-Bin; Zheng, Lan-Sun

2013-09-01

Four Zn(II) coordination complexes, namely {[Zn(pmbm)2(tpa)]·H2O}n (1), {[Zn(pmbm)(phda)]·2(H2O)}n (2), [Zn(pmbm)(aze)]n (3), {[Zn(pmbm)(1,4-ndc)]·2(CH3OH)}n (4) [pmbm = 1-(4-pyridylmethyl)-benzimidazole, H2tpa = terephthalic acid, H2phda = phenylenediacetic acid, H2aze = azelaic acid, 1,4-ndcH2 = 1,4-naphthalenedicarboxylic acid] have been synthesized by solution phase ultrasonic reactions of Zn(AC)2·2H2O with pmbm and various dicarboxylates ligands under the ammoniacal condition. All the complexes have been characterized by elemental analyses, IR spectra and X-ray diffraction. Complexes 1 and 2 exhibit one-dimensional chains structure and complex 3 and 4 are two-dimensional sheets structure with (4,4) topology. Complexes 1-4 spanning from one-dimensional chains to two-dimensional sheets suggest that dicarboxylates play significant roles in the formation of such coordination architectures. The photoluminescences of the complexes were also investigated in the solid state at room temperature.

Col-F, a fluorescent probe for ex vivo confocal imaging of collagen and elastin in animal tissues.

PubMed

Biela, Ewa; Galas, Jerzy; Lee, Brian; Johnson, Gary L; Darzynkiewicz, Zbigniew; Dobrucki, Jurek W

2013-06-01

A new low-molecular-weight fluorescent probe, Col-F, that exhibits affinity to collagen and elastin, was used successfully in imaging of extracellular matrix in freshly excised animal tissues. Col-F readily penetrates between live cells into tissues and binds to fibers of collagen and elastin by a noncovalent mechanism. Fibers of collagen and elastin have been stained in a variety of tissues, including tendon, skeletal muscle, connective tissue, and arteries. Cells migrating in a Col-F-stained collagenous biomaterial were also imaged. No phototoxic effects were detected when live keratocytes were imaged in the in vitro culture in the presence of Col-F. In conclusion, Col-F provides a simple and convenient tool for fluorescence three-dimensional imaging of intricate collagenous and elastic structures in live and fixed animal tissues, as well as in collagen-containing biomaterials. Copyright © 2013 International Society for Advancement of Cytometry.

Superlinear variant of the dual affine scaling algorithm

DOE Office of Scientific and Technical Information (OSTI.GOV)

Luz, C.; Cardosa, D.

1994-12-31

The affine scaling methods introduced by Dikin are generally considered the most efficient interior point algorithms from a computational point of view. However, it is actually an open question to know whether there is a polynomial affine scaling algorithm. This fact has motivated many investigations efforts and led to several convergence results. This is the case of the recently obtained results by Tsuchiya, Tseng and Luo and Tsuchiya and Muramatsu which, unlike the pioneering Dikin`s convergence result, do not require any non degeneracy assumption. This paper presents a new variant of the dual affine scaling algorithm for Linear Programming that,more » in a finite number of iterations, determines a primal-dual pair of optimal solutions. It is also shown the superlinear convergence of that variant without requiring any non degeneracy assumption.« less

Facile Affinity Maturation of Antibody Variable Domains Using Natural Diversity Mutagenesis

PubMed Central

Tiller, Kathryn E.; Chowdhury, Ratul; Li, Tong; Ludwig, Seth D.; Sen, Sabyasachi; Maranas, Costas D.; Tessier, Peter M.

2017-01-01

The identification of mutations that enhance antibody affinity while maintaining high antibody specificity and stability is a time-consuming and laborious process. Here, we report an efficient methodology for systematically and rapidly enhancing the affinity of antibody variable domains while maximizing specificity and stability using novel synthetic antibody libraries. Our approach first uses computational and experimental alanine scanning mutagenesis to identify sites in the complementarity-determining regions (CDRs) that are permissive to mutagenesis while maintaining antigen binding. Next, we mutagenize the most permissive CDR positions using degenerate codons to encode wild-type residues and a small number of the most frequently occurring residues at each CDR position based on natural antibody diversity. This mutagenesis approach results in antibody libraries with variants that have a wide range of numbers of CDR mutations, including antibody domains with single mutations and others with tens of mutations. Finally, we sort the modest size libraries (~10 million variants) displayed on the surface of yeast to identify CDR mutations with the greatest increases in affinity. Importantly, we find that single-domain (VHH) antibodies specific for the α-synuclein protein (whose aggregation is associated with Parkinson’s disease) with the greatest gains in affinity (>5-fold) have several (four to six) CDR mutations. This finding highlights the importance of sampling combinations of CDR mutations during the first step of affinity maturation to maximize the efficiency of the process. Interestingly, we find that some natural diversity mutations simultaneously enhance all three key antibody properties (affinity, specificity, and stability) while other mutations enhance some of these properties (e.g., increased specificity) and display trade-offs in others (e.g., reduced affinity and/or stability). Computational modeling reveals that improvements in affinity are generally

Nanostructured biosensor for detecting glucose in tear by applying fluorescence resonance energy transfer quenching mechanism.

PubMed

Chen, Longyi; Tse, Wai Hei; Chen, Yi; McDonald, Matthew W; Melling, James; Zhang, Jin

2017-05-15

In this paper, a nanostructured biosensor is developed to detect glucose in tear by using fluorescence resonance energy transfer (FRET) quenching mechanism. The designed FRET pair, including the donor, CdSe/ZnS quantum dots (QDs), and the acceptor, dextran-binding malachite green (MG-dextran), was conjugated to concanavalin A (Con A), an enzyme with specific affinity to glucose. In the presence of glucose, the quenched emission of QDs through the FRET mechanism is restored by displacing the dextran from Con A. To have a dual-modulation sensor for convenient and accurate detection, the nanostructured FRET sensors were assembled onto a patterned ZnO nanorod array deposited on the synthetic silicone hydrogel. Consequently, the concentration of glucose detected by the patterned sensor can be converted to fluorescence spectra with high signal-to-noise ratio and calibrated image pixel value. The photoluminescence intensity of the patterned FRET sensor increases linearly with increasing concentration of glucose from 0.03mmol/L to 3mmol/L, which covers the range of tear glucose levels for both diabetics and healthy subjects. Meanwhile, the calibrated values of pixel intensities of the fluorescence images captured by a handhold fluorescence microscope increases with increasing glucose. Four male Sprague-Dawley rats with different blood glucose concentrations were utilized to demonstrate the quick response of the patterned FRET sensor to 2µL of tear samples. Copyright © 2016 Elsevier B.V. All rights reserved.

2D Affine and Projective Shape Analysis.

PubMed

Bryner, Darshan; Klassen, Eric; Huiling Le; Srivastava, Anuj

2014-05-01

Current techniques for shape analysis tend to seek invariance to similarity transformations (rotation, translation, and scale), but certain imaging situations require invariance to larger groups, such as affine or projective groups. Here we present a general Riemannian framework for shape analysis of planar objects where metrics and related quantities are invariant to affine and projective groups. Highlighting two possibilities for representing object boundaries-ordered points (or landmarks) and parameterized curves-we study different combinations of these representations (points and curves) and transformations (affine and projective). Specifically, we provide solutions to three out of four situations and develop algorithms for computing geodesics and intrinsic sample statistics, leading up to Gaussian-type statistical models, and classifying test shapes using such models learned from training data. In the case of parameterized curves, we also achieve the desired goal of invariance to re-parameterizations. The geodesics are constructed by particularizing the path-straightening algorithm to geometries of current manifolds and are used, in turn, to compute shape statistics and Gaussian-type shape models. We demonstrate these ideas using a number of examples from shape and activity recognition.

Detection of adenosine triphosphate in HeLa cell using capillary electrophoresis-laser induced fluorescence detection based on aptamer and graphene oxide.

PubMed

Fang, Bi-Yun; Yao, Ming-Hao; Wang, Chun-Yuan; Wang, Chao-Yang; Zhao, Yuan-Di; Chen, Fang

2016-04-01

A method for ATP quantification based on dye-labeled aptamer/graphene oxide (aptamer/GO) using capillary electrophoresis-laser induced fluorescence (CE-LIF) detecting technique has been established. In this method, the carboxyfluorescein (FAM)-labelled ATP aptamers were adsorbed onto the surface of GO, leading to the fluorescence quenching of FAM; after the incubation with a limited amount of ATP, stronger affinity between ATP aptamer and ATP resulted in the desorption of aptamers and the fluorescence restoration of FAM. Then, aptamer-ATP complex and excess of aptamer/GO and GO were separated and quantified by CE-LIF detection. It was shown that a linear relation was existing in the CE-LIF peak intensity of aptamer-ATP and ATP concentration in range of 10-700 μM, the regression equation was F=1.50+0.0470C(ATP) (R(2)=0.990), and the limit of detection was 1.28 μM (3S/N, n=5), which was one order magnitude lower than that of detection in solution by fluorescence method. The approach with excellent specificity and reproducibility has been successfully applied to detecting concentration of ATP in HeLa cell. Copyright © 2015 Elsevier B.V. All rights reserved.

Direct visualization of nucleolar G-quadruplexes in live cells by using a fluorescent light-up probe.

PubMed

Zhang, Suge; Sun, Hongxia; Chen, Hongbo; Li, Qian; Guan, Aijiao; Wang, Lixia; Shi, Yunhua; Xu, Shujuan; Liu, Meirong; Tang, Yalin

2018-05-01

Direct detection of G-quadruplexes in human cells has become an important issue due to the vital role of G-quadruplex related to biological functions. Despite several probes have been developed for detection of the G-quadruplexes in cytoplasm or whole cells, the probe being used to monitor the nucleolar G-quadruplexes is still lacking. Formation of the nucleolar G-quadruplex structures was confirmed by using circular dichroism (CD) spectroscopy. The binding affinity and selectivity of Thioflavin T (ThT) towards various DNA/RNA motifs in solution and gel system were measured by using fluorescence spectroscopy and polyacrylamide gel electrophoresis (PAGE), respectively. G-quadruplex imaging in live cells was directly captured by using confocal laser scanning microscopy (CLSM). Formation of the rDNA and rRNA G-quadruplex structures is demonstrated in vitro. ThT is found to show much higher affinity and selectivity towards these G-quadruplex structures versus other nucleic acid motifs either in solution or in gel system. The nucleolar G-quadruplexes in living cells are visualized by using ThT as a fluorescent probe. G-quadruplex-ligand treatments in live cells lead to sharp decrease of ThT signal. The natural existence of the G-quadruplexes structure in the nucleoli of living cells is directly visualized by using ThT as an indicator. The research provides substantive evidence for formation of the rRNA G-quadruplex structures, and also offers an effective probe for direct visualization of the nucleolar G-quadruplexes in living cells. Copyright © 2018. Published by Elsevier B.V.

Protein labeling for live cell fluorescence microscopy with a highly photostable renewable signal† †Electronic supplementary information (ESI) available: Supplementary methods, figures, movies, and data. See DOI: 10.1039/c7sc01628j

PubMed Central

Bozhanova, Nina G.; Baranov, Mikhail S.; Klementieva, Natalia V.; Sarkisyan, Karen S.; Gavrikov, Alexey S.; Yampolsky, Ilia V.; Zagaynova, Elena V.; Lukyanov, Sergey A.; Lukyanov, Konstantin A.

2017-01-01

We present protein-PAINT – the implementation of the general principles of PAINT (Point Accumulation for Imaging in Nanoscale Topography) for live-cell protein labeling. Our method employs the specific binding of cell-permeable fluorogenic dyes to genetically encoded protein tags. We engineered three mutants of the bacterial lipocalin Blc that possess different affinities to a fluorogenic dye and exhibit a strong increase in fluorescence intensity upon binding. This allows for rapid labeling and washout of intracellular targets on a time scale from seconds to a few minutes. We demonstrate an order of magnitude higher photostability of the fluorescence signal in comparison with spectrally similar fluorescent proteins. Protein-PAINT ensures prolonged super-resolution fluorescence microscopy of living cells in both single molecule detection and stimulated emission depletion regimes. PMID:29147545

Local uncontrollability for affine control systems with jumps

NASA Astrophysics Data System (ADS)

Treanţă, Savin

2017-09-01

This paper investigates affine control systems with jumps for which the ideal If(g1, …, gm) generated by the drift vector field f in the Lie algebra L(f, g1, …, gm) can be imbedded as a kernel of a linear first-order partial differential equation. It will lead us to uncontrollable affine control systems with jumps for which the corresponding reachable sets are included in explicitly described differentiable manifolds.

Understanding Zinc Quantification with Existing and Advanced Ditopic Fluorescent Zinpyr Sensors

PubMed Central

Buccella, Daniela; Horowitz, Joshua A.; Lippard, Stephen J.

2011-01-01

Treatment of aqueous zinc solutions with incremental additions of a ditopic fluorescent sensor of the Zinpyr family, based on pyridine/pyrazine-containing metal recognition units, affords a fluorescence titration curve with a sharp maximum at a sensor:Zn2+ ratio of 0.5 (Zhang, X-a.; Hayes, D.; Smith, S. J.; Friedle, S.; Lippard, S. J. J. Am. Chem Soc. 2008, 130, 15788–15789). This fluorescence response enables the quantification of readily chelatable zinc in biological samples by a simple titration protocol. In the present work a new set of ditopic fluorescence zinc sensors functionalized with pyridine/pyrazine-containing metal chelating units is described, and through detailed studies the principles governing the characteristic “OFF-ON-OFF” fluorescence behavior and quantification capabilities of the family are delineated. Incorporation of carboxylate/ester groups in the 6 position of the fluorescein allows for control of the spatial distribution of the sensor for selective extra- or intracellular imaging of mobile zinc, without introducing significant changes in zinc-binding properties. A combination of spectrophotometric and potentiometric measurements provided a complete description of the H+ and Zn2+ binding properties of the compounds and their correlation with the observed fluorescence profile. The first zinc-binding event has an apparent affinity, K1′, of 1.9–3.1×109 M−1, whereas for coordination of the second Zn2+ ion, responsible for fluorescence turn on, the apparent formation constant K2′ is 5.5–6.9×107 M−1. A detailed chemical and mathematical analysis of the system demonstrated that the difference in emission efficiencies of the dimetalated (LZn2) vs. monometalated (LZn) and metal free (L) forms, a consequence of the combined quenching effects of the two metal-chelating units, significantly influences the shape of the titration curve. The scope of the titration method was investigated mathematically, and a lower boundary for the

Fluorescence turn-on detection of iodide, iodate and total iodine using fluorescein-5-isothiocyanate-modified gold nanoparticles.

PubMed

Chen, Yi-Ming; Cheng, Tian-Lu; Tseng, Wei-Lung

2009-10-01

Selective turn-on fluorescence detection of I(-) was accomplished using fluorescein isothiocyanate-decorated gold nanoparticles (FITC-AuNPs). FITC molecules, which fluoresce strongly in an alkaline solution, were severely quenched when they were attached to the surface of AuNPs through their isothiocyanate group. Upon the addition of I(-), FITC molecules were detached because of I(-) adsorption on the surface of AuNPs. As a result, released FITC molecules were restored to their original fluorescence intensity. Because I(-) has a higher binding affinity to the surface of Au than do Br(-), Cl(-), or F(-), the FITC-AuNPs obviously have a higher selectivity toward I(-) than toward these other anions. Meanwhile, after IO(3)(-) was reduced to I(-) with ascorbic acid, the detection of IO(3)(-) was successfully achieved using the FITC-AuNPs. Under an optimum pH and AuNP concentration, the lowest detectable concentrations of I(-) and IO(3)(-) using this probe were 10.0 and 50.0 nM, respectively. The FITC-AuNPs provide a number of advantages, including easy preparation, selectivity, sensitivity, and low cost. This unique probe was applied to an analysis of the total iodine in edible salt and seawater.

Malachite green mediates homodimerization of antibody VL domains to form a fluorescent ternary complex with singular symmetric interfaces

PubMed Central

Szent-Gyorgyi, Chris; Stanfield, Robyn L.; Andreko, Susan; Dempsey, Alison; Ahmed, Mushtaq; Capek, Sara; Waggoner, Alan; Wilson, Ian A.; Bruchez, Marcel P.

2013-01-01

We report that a symmetric small molecule ligand mediates the assembly of antibody light chain variable domains (VLs) into a correspondent symmetric ternary complex with novel interfaces. The L5* Fluorogen Activating Protein (FAP) is a VL domain that binds malachite green dye (MG) to activate intense fluorescence. Crystallography of liganded L5* reveals a 2:1 protein:ligand complex with inclusive C2 symmetry, where MG is almost entirely encapsulated between an antiparallel arrangement of the two VL domains. Unliganded L5* VL domains crystallize as a similar antiparallel VL/VL homodimer. The complementarity determining regions (CDRs) are spatially oriented to form novel VL/VL and VL/ligand interfaces that tightly constrain a propeller conformer of MG. Binding equilibrium analysis suggests highly cooperative assembly to form a very stable VL/MG/VL complex, such that MG behaves as a strong chemical inducer of dimerization. Fusion of two VL domains into a single protein tightens MG binding over 1,000-fold to low picomolar affinity without altering the large binding enthalpy, suggesting that bonding interactions with ligand and restriction of domain movements make independent contributions to binding. Fluorescence activation of a symmetrical fluorogen provides a selection mechanism for the isolation and directed evolution of ternary complexes where unnatural symmetric binding interfaces are favored over canonical antibody interfaces. As exemplified by L5*, these self-reporting complexes may be useful as modulators of protein association or as high affinity protein tags and capture reagents. PMID:23978698

Fluorescent and Magnetic Mesoporous Hybrid Material: A Chemical and Biological Nanosensor for Hg2+ Ions

PubMed Central

Suresh, Moorthy; Anand, Chokkalingam; Frith, Jessica E.; Dhawale, Dattatray S.; Subramaniam, Vishnu P.; Strounina, Ekaterina; Sathish, Clastinrusselraj I.; Yamaura, Kazunari; Cooper-White, Justin J.; Vinu, Ajayan

2016-01-01

We introduce “sense, track and separate” approach for the removal of Hg2+ ion from aqueous media using highly ordered and magnetic mesoporous ferrosilicate nanocages functionalised with rhodamine fluorophore derivative. These functionalised materials offer both fluorescent and magnetic properties in a single system which help not only to selectively sense the Hg2+ ions with a high precision but also adsorb and separate a significant amount of Hg2+ ion in aqueous media. We demonstrate that the magnetic affinity of these materials, generated from the ultrafine γ-Fe2O3 nanoparticles present inside the nanochannels of the support, can efficiently be used as a fluorescent tag to sense the Hg2+ ions present in NIH3T3 fibroblasts live cells and to track the movement of the cells by external magnetic field monitored using confocal fluorescence microscopy. This simple approach of introducing multiple functions in the magnetic mesoporous materials raise the prospect of creating new advanced functional materials by fusing organic, inorganic and biomolecules to create advanced hybrid nanoporous materials which have a potential use not only for sensing and the separation of toxic metal ions but also for cell tracking in bio-separation and the drug delivery. PMID:26911660

Exciton-controlled fluorescence: application to hybridization-sensitive fluorescent DNA probe.

PubMed

Okamoto, Akimitsu; Ikeda, Shuji; Kubota, Takeshi; Yuki, Mizue; Yanagisawa, Hiroyuki

2009-01-01

A hybridization-sensitive fluorescent probe has been designed for nucleic acid detection, using the concept of fluorescence quenching caused by the intramolecular excitonic interaction of fluorescence dyes. We synthesized a doubly thiazole orange-labeled nucleotide showing high fluorescence intensity for a hybrid with the target nucleic acid and effective quenching for the single-stranded state. This exciton-controlled fluorescent probe was applied to living HeLa cells using microinjection to visualize intracellular mRNA localization. Immediately after injection of the probe into the cell, fluorescence was observed from the probe hybridizing with the target RNA. This fluorescence rapidly decreased upon addition of a competitor DNA. Multicoloring of this probe resulted in the simple simultaneous detection of plural target nucleic acid sequences. This probe realized a large, rapid, reversible change in fluorescence intensity in sensitive response to the amount of target nucleic acid, and facilitated spatiotemporal monitoring of the behavior of intracellular RNA.

Quantifying domain-ligand affinities and specificities by high-throughput holdup assay

PubMed Central

Vincentelli, Renaud; Luck, Katja; Poirson, Juline; Polanowska, Jolanta; Abdat, Julie; Blémont, Marilyne; Turchetto, Jeremy; Iv, François; Ricquier, Kevin; Straub, Marie-Laure; Forster, Anne; Cassonnet, Patricia; Borg, Jean-Paul; Jacob, Yves; Masson, Murielle; Nominé, Yves; Reboul, Jérôme; Wolff, Nicolas; Charbonnier, Sebastian; Travé, Gilles

2015-01-01

Many protein interactions are mediated by small linear motifs interacting specifically with defined families of globular domains. Quantifying the specificity of a motif requires measuring and comparing its binding affinities to all its putative target domains. To this aim, we developed the high-throughput holdup assay, a chromatographic approach that can measure up to a thousand domain-motif equilibrium binding affinities per day. Extracts of overexpressed domains are incubated with peptide-coated resins and subjected to filtration. Binding affinities are deduced from microfluidic capillary electrophoresis of flow-throughs. After benchmarking the approach on 210 PDZ-peptide pairs with known affinities, we determined the affinities of two viral PDZ-binding motifs derived from Human Papillomavirus E6 oncoproteins for 209 PDZ domains covering 79% of the human PDZome. We obtained exquisite sequence-dependent binding profiles, describing quantitatively the PDZome recognition specificity of each motif. This approach, applicable to many categories of domain-ligand interactions, has a wide potential for quantifying the specificities of interactomes. PMID:26053890

Affinity study on bovine serum albumin's peptides to amphiphilic gold nanoparticles: A test of epitopes and non-epitopes

NASA Astrophysics Data System (ADS)

Yuan, Ming; Li, Wanrong; Yang, Mingming; Huang, Xiufeng; Bai, Zhijun; Liu, Yushuang; Cai, Weijun; Wang, Yuqin; Zhang, Feng

2017-09-01

It is an inevitable event that nanoparticles (NPs) will encounter proteins/peptides in nano-medicine, so it has been significant to know their interaction mechanism before in vivo applications. Previously, a 105-amino-acid sequence had been reported as the binding site between bovine serum albumin (BSA) and amphiphilic polymer coated gold nanoparticles (AP-AuNPs) along with a mortise-tenon joint hypothesis. This article tested the affinity difference between two epitope peptide sequences such as: LGEYGFQNALIVR (S1), DAFLGSFLYEYSR (S2) and one non-epitope peptide sequence as: FDEHVKLVNELTEF (S3). With the photoluminescent amino acid residues, the fluorescence quenching method based on the nanometal surface energy transfer (NSET) principle was able to study the thermodynamics of the current binding system. The binding constants (Ka) were determined and followed the order as: Ka-S1 > Ka-S2 >> Ka-S3. Moreover, Hill constants indicated that cooperativity only presented in the interactions of AP-AuNP with either S1 or S2, but not for S3. Moreover, gel electrophoresis, surface plasmon resonance, atomic force microscopy and three dimensional fluorescence microscopy were all also used to comprehensively analyse the binding interaction mechanism. These results further provided useful information to better understand the mortise-tenon joint, which might find applications to nanofabrication and biomedicine.